Isobaric Multiplex Reagents for Carbonyl Containing Compound High-Throughput Quantitative Analysis

ABSTRACT

The present invention provides a set of novel isobaric chemical tags, also referred herein as SUGAR (Isobaric Multiplex Reagents for Carbonyl Containing Compound). These labeling tags are compact and easy to synthesize at high yield and purity in just a few steps using commercially available starting materials. The tagging reagents of the present invention comprise: a) a reporter group, having at least one atom that is optionally isotopically labeled; b) a balancing group, also having at least one atom that is optionally isotopically labeled, and c) an aldehyde, ketone, or carboxylic acid reactive group. The multiplex SUGAR tags are able to react with an aldehyde, ketone, or carboxylic acid group of the molecule to be tagged, which offers the capability for labeling and quantitation of glycans, proteins/peptides, and fatty acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. patent application Ser. No.16/252,464, filed Jan. 18, 2019, which claims priority from U.S.Provisional Patent Application No. 62/619,414, filed Jan. 19, 2018,which is incorporated by reference herein to the extent that there is noinconsistency with the present disclosure.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under AG055377 andDK071801 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Quantitative measurements of proteins and small molecules are essentialto understanding biological systems and disease mechanisms. For example,proteomics is a systematic study that aims to comprehensivelycharacterize and quantify proteins in a biological system. Massspectrometry (MS) is the most common tool in proteomic analysis.

There are two main types of quantification strategies in such studies.One of them is label free quantification. The extracted molecules (e.g.,proteins) are digested by an enzyme, separated by chromatographic orelectrophoretic system, followed by detection on a mass spectrometer foridentification and quantitation. The database searching engine comparesthe theoretical spectra and experimental spectra to identify peptidesand proteins. The quantification result is generated by comparing theintensity of every species in different samples. For label freequantification strategy, the sample preparation is relativelystraightforward and less time consuming while the MS-based dataacquisition would require more instrument time since only one samplecould be analyzed in a single LCMS experiment.

The alternative strategy is chemical labeling prior to the LCMSanalysis. The molecules (e.g., protein analytes) are enzymaticallydigested, labeled with chemical tags, purified, separated and analyzedby an LCMS system. The identification process is almost the same withaddition of derivative as a fixed chemical modification. Thequantification result is produced by comparing either the intensities ofsignature ions or peak areas under curve resulting from differentisotopic labeling tags.

The most commonly used chemical labeling is isobaric labeling because ofthe high plex capacity, simple spectral identification and accuraterelative quantification. There are several chemical derivatizationreagents for proteomics including TMT, iTRAQ and DiLeu-based tags. Suchconventional tags react with primary amines at the N-terminus ofpeptides or lysine side chains, although several reagents have beendeveloped for C-terminal derivatization. Additional isobaric taggingreagents containing amine reactive groups and enabling 8 plex and 16plex analysis were also developed and described in U.S. Pat. No.9,388,132.

However, limited success has been achieved for quantitative analysis ofC-terminally labeled peptides via isobaric tagging strategy. Since thecharge state of C-terminally labeled peptides could be increasedefficiently compared to negatively charged carboxylic acid without alabel, the labile post-translational modifications (PTMs) analysis withelectron-transfer dissociation (ETD) or electron-transfer/higher-energycollision dissociation (EThcD) fragmentation would be beneficial withC-terminal derivatization. Higher charge state peptides would producebetter fragmentation via ETD or EThcD fragmentation with confidentidentification and better sequence coverage.

With technological advancement of MS, MS-based quantitative glycomicshas also become promising. The backbone fragments produced during MSfragmentation can be used to elucidate structure for characterizationwhile intensities of parent ions or fragment peaks enable relativequantification of glycans.

MS-based stable isotope labeling remains a key technology to quantifyproteins, glycans, small molecules, and metabolites, where stable heavyisotopes can be differentially incorporated into analytes chemically(e.g., labeling tags) or metabolically (e.g., SILAC). Compared tolabel-free approaches, stable isotope labeling allows for simultaneouscomparison of multiple samples in a single MS run (multiplexing) withimproved accuracy and reduced systematic variation for quantitativeproteomics and metabolomics.

Glycans (or carbohydrates) are ubiquitous in biological system and areinvolved in a wide range of biological functions, including proteinfolding, cell adhesion and trafficking, cell signaling, fertilizationand embryogenesis; and pathogen recognition and immune responses.Furthermore, glycosylation is one of the most importantpost-translational modifications of peptides and is involved in severalbiological processes, including cell-cell recognition, communication,and immunity response. Abnormal glycosylation has been implicated in anumber of diseases, including cancer, cardiovascular disease, andimmunological disorders.

Glycans are highly complex entities with multiple building units anddifferent degrees of branched polymerization. Intensive research effortshave been directed to mass spectrometry (MS)-based qualitative andquantitative glycomic analysis due to important functions of glycans.However, the complexity of carbohydrates, which is amplified by thepresence of stereo-isomers, anomeric configurations, branched chains,and modifications such as sulfation, methylation, and phosphorylation,render study of the biological roles of glycans intractable to mostbiomedical researchers. Compared to genomics and protein biochemistry,glycoscience suffers from the inability to carry out high throughputsynthesis or structural and functional analysis of glycans.

As a result, researchers have attempted to develop isobaric chemicaltags for glycan MS analysis. Unfortunately, the performance of thesetags has left a lot to be desired. For example, AminoxyTMT, a set ofcommercially available tags from Thermo Scientific, suffers from limitedreporter ion yield and poor labeling efficiency for complex glycans.Thus, there is a need for a set of improved tags that address theselimitations.

SUMMARY OF THE INVENTION

To overcome the limitations of existing isobaric chemical tags, thepresent invention provides improved compositions and methods of labelingglycans and other molecules using novel isobaric tandem massspectrometry (MS²) tagging reagents with high quantitation efficacy andgreatly reduced cost for proteomics, glycomics, glycomic quantitation,and small molecule quantification. In particular, the present inventionprovides isobaric labeling tags useful for the analysis of moleculescontaining aldehyde, ketone, or carboxylic acid groups. In certainaspects, the present invention provides the design and synthesis of aset of novel amino acid and N, N-dimethylated amino acid-based isobaric8-plex (or greater) reagents, 12-plex (or greater) reagents, as well as16-plex (or greater) reagents, able to bind to aldehyde, ketone, andcarboxylic acid groups. Preliminary data of synthesis and glycanlabeling is also presented.

In an embodiment, the present invention provides a set of novel isobaricchemical tagging reagents, coined SUGAR (Isobaric Multiplex Reagents forCarbonyl Containing Compound), that are compact and easy to synthesizeat high yield (greater than 50%) and purity in just a few steps usingcommercially available starting materials as generally shown in Scheme 1below:

The multiplex SUGAR tags are both aldehyde-reactive and carboxylicacid-reactive, offering the capability for labeling and quantitation ofglycans, proteins/peptides, and fatty acids. For 4-plex SUGAR tags, thereporter ions with 1 Da mass difference in MS² spectra allow the use ofmass spectrometers with modest resolution (resolving power <60K), makingthem compatible with a variety of instrument platforms and accessible toa larger number of users.

The tagging reagents of the present invention comprise: a) a reportergroup, having at least one atom that is optionally isotopically labeled;b) a balancing group, also having at least one atom that is optionallyisotopically labeled, and c) an aldehyde, ketone, or carboxylic acidreactive group able to react with an aldehyde, ketone, or carboxylicacid group of the molecule to be tagged, such as a glycosylated sidechain of a peptide.

A major limiting factor of previous isobaric tagging reagents is thatthey are limited to use with molecules having amine groups and areunable to react with carboxylic acid groups, or similar reactive groups,present in glycans. For example, activated carboxylic acid groups inprevious tagging reagents cannot react with the reducing end of a glycanto form the conjugates for MS analysis. Similarly, previous isobarictagging reagents are limited in the number of atoms able to beisotopically labeled in the balancing group. For example, previouslyreported dimethylated leucine (DiLeu) 4-plex reagents able to react toamine groups of peptides utilize isotopic carbonyl groups, which onlycontain two atoms. As a result, only four isotopic combinations can beachieved within the balancing group. The isobaric tagging reagents ofthe present invention have balancing groups able to provide a greaternumber of isotopic combinations. As a result, the present inventionprovides compositions and methods of tagging peptides and othermolecules using, at least, 8-plex, 12-plex, and 16-plex isobaric tandemmass tagging reagents, including novel N, N-dimethylated amino acidbased 8-plex, 12-plex, and 16-plex tagging reagents.

In one embodiment, multiple tagging reagents are used to label two ormore molecules having at least one aldehyde, ketone, or carboxylic acidgroup, or a mixture of such molecules, wherein the tagging reagents havethe same molecular weight as one another, but wherein the reporter groupof each tagging reagent has a different mass due to the differentisotopically labeled atoms in each reporter group. Similarly, thebalancing group of each tagging reagent has a different mass from oneanother due to the different isotopically labeled atoms in eachbalancing group. In one embodiment, each molecule sample is labeledindividually, pooled together, and introduced into the mass spectrometerfor quantitative analysis. Since the tagging reagents have the samemass, the labeled molecules will produce a single peak in MS mode, butupon MS² fragmentation, each sample labeled with a different taggingreagent will produce a unique reporter ion due to the mass differencebetween the reporter groups. Preferably, a molecule labeled with atagging reagent of the present invention is able to form a strongimmonium ion during MS² fragmentation.

The tagging reagents of the present invention can be used to label andquantify a wide range of molecules provided that the molecule containsan aldehyde, ketone, or carboxylic acid group able to react with thereactive group of the tagging reagent. For example, the molecules ableto be labeled using the compounds described herein include, but are notlimited to, glycosylated peptides, lipids, and other biologicalcompounds. In an embodiment, a target biological molecule in two or moresamples is labeled and subsequently analyzed using the tagging reagentsof the present invention, where at least one sample is a biologicalsample taken from a patient before a treatment is administered to thepatient, and one or more samples are biological samples taken from thepatient at one or more time periods after the treatment has beenadministered to the patient. In this way, the present invention can beused to determine if a treatment results in the increased or decreasedpresence of a biological molecule in the patient as a result of thetreatment. Optionally, the patient is a cancer patient and the treatmentis an anti-cancer treatment, such as chemotherapy or radiation therapy.The sample taken from the patient may include, but is not limited to, afluid sample (such as blood), cell sample, or tissue sample (e.g.,tissue biopsy). In an embodiment, the treatment is the administration ofa drug or therapeutic which may result in the increase or decrease of abiological molecule or metabolite.

In one embodiment, the SUGAR tagging reagents of the present inventionare derived from a dipeptide comprising two amino acids. Preferably theamino acids are natural amino acids, but the present inventioncontemplates the use of unnatural, non-standard and synthetic aminoacids, such as p amino acids, as the amino acid which makes up thereporter group, the balancing group, or both. In a further embodiment,the tagging reagents of the present invention are derived from adipeptide where the amino group of one amino acid has been dimethylated.During MS² fragmentation, the dipeptide will fragment to form a reporterion, preferably an immonium ion, which can be readily detected. In afurther embodiment, the SUGAR tagging reagents are derived fromN,N-dimethyl leucine (DiLeu); N,N-dimethyl isoleucine (Dille);N,N-dimethyl alanine (DiAla); N,N-dimethyl glycine (DiGly); N,N-dimethylvaline (DiVal); N,N-dimethyl histidine (DiHis); N,N-dimethylphenylalanine (DiPhe); N,N-dimethyl tryptophan (DiTrp); N,N-dimethyllysine (DiLys) or N,N-dimethyl tyrosine (DiTyr).

DiLeu derivative tandem mass tags show improved reporter ion yield andlabeling efficiency compared to the current commercially available tags.Low relative error (<15%) and standard deviation prove the excellentquantification accuracy and precision. DiLeu derivative tags are highlypromising for glycomic analysis in complex biological systems and can bepowerful tools to study glycosylation patterns in diseases.

As an example, a series of isobaric tagging reagents may comprise areporter group (which forms the reporter ion during fragmentation), abalance group, and an aldehyde, ketone, or carboxylic acid reactivegroup as shown below in Scheme 2 (using N,N-dimethyl leucine and alanineas the amino acids). One or more atoms in the reporter group, balancinggroup, or both, in each reagent are the isotopically heavy versions ofthe atom. Each tagging reagent in the series will have a differentcombination of atoms that are the isotopically heavy versions of theatoms, but with the condition that the total aggregate mass of eachtagging reagent is the same as the other tagging reagents in the series.For example, the nitrogen atom in the reporter group shown in Scheme 2may be ¹⁵N, one or more carbon atoms may be ¹³C, and one or morehydrogen atoms may be deuterium (D). This provides at least eightpossible reporter groups each having a different mass, including onereporter group containing no heavy isotopes. The balancing groups ofeach reagent will contain the appropriate number of heavy isotopes, suchas ¹⁸O, ¹³C, ¹⁵N or D, so that the combined mass of the balancing groupand reporter group are the same for each reagent. Varying the atomswhich contain the heavy isotope form in the reporter groups andbalancing groups allows each tagging reagent to have the same combinedmass but a different mass of the reporter group after fragmentation.

While the SUGAR tags illustrated in Schemes 1 and 2 have an aldehyde,ketone, or carboxylic acid reactive group which is a hydrazide, thereactive group can be any functional group able to react with analdehyde, ketone, or carboxylic acid group of a molecule thereby formingbond between the molecule and the balancing group of the taggingreagent. In an embodiment, the reactive group includes, but is notlimited to, a hydrazide, hydrazine, amine, or oxyamine.

The tagging reagents are reacted with one or more samples containing amolecule of interest containing an aldehyde, ketone, or carboxylic acidreactive group, such as a glycosylated peptide. The samples may then becombined together or with a known standard labeled with one of thetagging reagents. The combined sample is then analyzed using massspectrometry. After fragmentation, the reporter group for each taggedpeptide or molecule will present a different mass due to the differentlyisotopically labeled atoms. By comparing the relative signal intensityof the detected reporter groups during tandem mass spectrometry, theamounts of each tagged molecule can be quantified, especially if a knownstandard is used as one of the tagged molecules.

In one embodiment, the tagging reagents (absent the aldehyde, ketone, orcarboxylic acid reactive group) preferably have a molecular mass betweenapproximately 125 and 400 Daltons, preferably between approximately 150and 350 Daltons, even more preferably between approximately 180 and 250Daltons. This allows the reporter group to have a large enough mass tobe readily detected during mass spectrometry analysis, while largertagging reagents may result in inefficient synthesis and labeling due tosteric hindrance.

Fragmentation may be achieved using a variety of methods including, butnot limited to, collision induced dissociation (CID), higher-energycollision dissociation (HCD), surface induced dissociation (SID), laserinduced dissociation (LID), electron capture dissociation (ECD),electron transfer dissociation (ETD), ultraviolet photo-dissociation(UVPD) or any combination of these methods or any equivalents known inthe art of tandem mass spectrometry. The molecule fragments are thendetected, identified and optionally quantified using methods as known inthe art.

Methods of Analyzing a Mixture Using at Least 8-Plex or 16-Plex TaggingReagents

In one embodiment, the present invention provides a method of analyzinga molecule having an amine group comprising the steps of: a) providingthe molecule; b) labeling the molecule with a compound having theformula of:

-   -   wherein,    -   R¹ is an aldehyde, ketone, or carboxylic acid reactive group;    -   R², R³, R⁴, R⁵ and R⁶, independently of one another, are        selected from the group consisting of hydrogen, deuterium,        branched and unbranched C₁ to C₁₂ alkyl groups, C₁ to C₁₂        cycloalkyl groups, C₁ to C₁₂ alkenyl groups, C₁ to C₁₂        cycloalkenyl groups, C₄ to C₁₂ aryl groups and C₄ to C₁₂        arylalkyl groups, wherein each of R², R³, R⁴, R⁵ and R⁶        optionally contain one or more ¹³C atoms and one or more        deuterium atoms;    -   C^(V) and C^(x), independently of one another, are ¹²C or ¹³C,    -   O^(U) and O^(y), independently of one another, are ¹⁶O or ¹⁸O;        and    -   N^(z) and N^(W), independently of one another, are ¹⁴N or ¹⁵N,        and        c) fragmenting the labeled molecule to generate an immonium ion        from the labeled molecule; and d) detecting and analyzing        fragments of the labeled molecule. In an embodiment, R² and R³,        independently of one another, are CH₃, ¹³CH₃, CDH₂, ¹³CDH₂,        CD₂H, ¹³CD₂H, CD₃ or ¹³CD₃. In an embodiment, at least one of R²        or R³ contains a deuterium atom, and N^(z) is ¹⁵N, or N^(W) is        ¹⁵N. In an embodiment, R⁶ is hydrogen or deuterium. Optionally,        R¹ comprises a hydrazide, hydrazine, amine, or oxyamine.

Preferably, R⁴ and R⁵ are selected from the group consisting ofhydrogen, deuterium, branched and unbranched C₁ to C₈ alkyl groups, C₁to C₈ cycloalkyl groups, C₁ to C₈ alkenyl groups, C₁ to C₈ cycloalkenylgroups, C₆ to C₁₂ aryl groups and C₆ to C₁₂ arylalkyl groups. In oneembodiment, R⁴ and R⁵ are selected from the group consisting ofhydrogen, deuterium, branched and unbranched C₁ to C₄ alkyl groups, C₁to C₄ cycloalkyl groups, and C₂ to C₄ alkenyl groups.

Labeling the molecule comprises the step of reacting the aldehyde,ketone, or carboxylic acid reactive group of the tagging reagent withthe aldehyde, ketone, or carboxylic acid group of the molecule. In oneembodiment, the molecule is a glycosylated peptide or molecule, and thelabeled molecule has the formula:

In one embodiment, the compound has the formula:

In a further embodiment, R⁵ is a methyl group optionally containing oneor more deuterium atoms and wherein the carbon is ¹²C or ¹³C; R⁵ ishydrogen or deuterium; or R⁵ is an isopropyl group optionally containingone or more deuterium atoms and one or more ¹³C atoms.

In a further embodiment, the compound is selected from:

In a further embodiment, the compound is selected from:

In a further embodiment, the compound is selected from:

In one embodiment, the compound has the formula:

In a further embodiment, R⁵ is a methyl group optionally containing oneor more deuterium atoms and wherein the carbon is ¹²C or ¹³C; R⁵ ishydrogen or deuterium; or R⁵ is a butyl group optionally containing oneor more deuterium atoms and one or more ¹³C atoms.

In a further embodiment, the compound is selected from:

In a further embodiment, the compound is selected from:

In a further embodiment, the compound is selected from:

In a further embodiment, the compound is selected from:

In one embodiment, the compound has the formula:

In a further embodiment, R⁵ is a methyl group optionally containing oneor more deuterium atoms and wherein the carbon is ¹²C or ¹³C; R⁵ is anisopropyl group optionally containing one or more deuterium atoms andone or more ¹³C atoms; or R⁵ is a butyl group optionally containing oneor more deuterium atoms and one or more ¹³C atoms.

In a further embodiment, the compound is selected from:

In a further embodiment, the compound is selected from:

In a further embodiment, the compound is selected from:

In a further embodiment, the compound is selected from:

In one embodiment, the compound has the formula:

In a further embodiment, R⁵ is hydrogen or deuterium; R⁵ is an isopropylgroup optionally containing one or more deuterium atoms and one or more¹³C atoms; or R⁵ is a butyl group optionally containing one or moredeuterium atoms and one or more ¹³C atoms.

In a further embodiment, the compound is selected from:

In a further embodiment, the compound is selected from:

In a further embodiment, the compound is selected from:

In a further embodiment, the compound is selected from:

In one embodiment, the compound has the formula:

In a further embodiment, R⁵ is hydrogen or deuterium; R⁵ is an isopropylgroup optionally containing one or more deuterium atoms and one or more¹³C atoms; or R⁵ is a methyl group optionally containing one or moredeuterium atoms and wherein the carbon is ¹²C or ¹³C.

In a further embodiment, the compound is selected from:

In a further embodiment, the compound is selected from:

In a further embodiment, the compound is selected from:

In the above embodiments, R², R³, R⁴, R⁵, R⁶, C^(x), C^(v), O^(y),O^(U), N^(z) and N^(W) are selected so that the mass of the reportergroup and balancing group for each tagging reagent applied to a mixtureare different while the overall mass of the tagging reagent remains thesame. In one embodiment, R² and R³ are CDH₂, CD₂H or CD₃. In a furtherembodiment, R² and R³ are CDH₂, N^(z) is ¹⁵N, and C^(x) is ¹³C. In afurther embodiment, R² and R³ are CDH₂ and O^(y) is ¹⁸O. In a furtherembodiment, N^(z) is ¹⁵N, C^(x) is ¹³C, and O^(y) is ¹⁸O.

While it is preferable for the balancing group of the tagging reagentsto contain an amino acid as described above, it is also possible toprovide tagging reagents that use other balancing groups as long as thebalancing group provides sufficient atoms able to be isotopicallylabeled in order to balance the isotopes in the reporter group andprovide the same aggregate mass for each of the tagging reagents. Oneembodiment of the invention provides methods of labeling targetmolecules using a tagging reagent having the formula:

wherein

R¹ is an aldehyde, ketone, or carboxylic acid reactive group,R², R³, R⁴, R⁵ and R⁶, independently of one another, are selected fromthe group consisting of hydrogen, deuterium, branched and unbranched C₁to C₁₂ alkyl groups, C₁ to C₁₂ cycloalkyl groups, C₁ to C₁₂ alkenylgroups, C₁ to C₁₂ cycloalkenyl groups, C₄ to C₁₂ aryl groups and C₄ toC₁₂ arylalkyl groups, wherein each of R², R³, R⁴ and R⁵ optionallycontain one or more ¹³C atoms and one or more deuterium atoms;C^(T) C^(V) and C^(x), independently of one another, are ¹²C or ¹³C,O^(y) is ¹⁶O or ¹⁸O; andN^(z) is ¹⁴N or ¹⁵N. Preferably, R² and R³, independently of oneanother, are CH₃, ¹³CH₃, CDH₂, ¹³CDH₂, CD₂H, ¹³CD₂H, CD₃ or ¹³CD₃.Preferably, R⁶ is hydrogen or deuterium. Optionally, R¹ comprises ahydrazide, hydrazine, amine, or oxyamine.

In one embodiment, the present invention provides a method of analyzinga mixture containing target molecules comprising the steps of: a)labeling target molecules within a first sample with a first taggingreagent, thereby generating first labeled target molecules; b) labelingtarget molecules within at least one additional sample with at least oneadditional tagging reagent, thereby generating additional labeled targetmolecules; c) combining the labeled target molecules of steps a) and b);d) fragmenting the combined labeled target molecules; and e) analyzingthe fragments of the labeled target molecules. The fragments can beanalyzed using mass spectrometry. Preferably, the fragmenting stepgenerates immonium ions from the labeled target molecules. Additionaltagging reagents having the same mass can be used to label molecules inadditional samples. The different samples are optionally combined andthe relative amounts of the tagged molecules compared. One of thesamples may be a glycosylated protein or molecule present in knownamount, allowing the quantitative amounts of target molecules from theother samples to be determined.

A further embodiment comprises labeling target molecules within at leasttwo additional samples with at least two additional tagging reagents;labeling target molecules within at least three additional samples withat least three additional tagging reagents; labeling target moleculeswithin at least four additional samples with at least four additionaltagging reagents; labeling target molecules within at least fiveadditional samples with at least five additional tagging reagents;labeling target molecules within at least six additional samples with atleast six additional tagging reagents; labeling target molecules withinat least seven additional samples with at least seven additional taggingreagents; and labeling target molecules within at least eight additionalsamples with at least eight additional tagging reagents.

The present invention provides 8-plex, 12-plex, and 16-plex taggingreagents able to produce reporter ions that differ by at least oneDalton from one another. Additionally, these tagging reagents can beused to provide 4-plex, 8-plex, and 12-plex tagging reagents whosereporter ions differ by two or more Daltons from one another. In afurther embodiment, the generated immonium ions of each tagging reagenthave a mass that differs from any of the other tagging reagents by twoor more Daltons.

In a further embodiment, each tagging reagent comprises a reportergroup, an aldehyde, ketone, or carboxylic acid reactive group, and abased balancing group located between the reporter group and thealdehyde, ketone, or carboxylic acid reactive group. One or more atomsin the reporter group, balancing group, or both, are isotopically heavyversions of the atom. The reporter group of each tagging reagent has amass different than the reporter groups of the other tagging reagents,the balancing group of each tagging reagent has a mass different thanthe balancing groups of other tagging reagents, and the aggregate massof the reporter groups plus the balancing group for each tagging reagentis the same.

In an embodiment, the balancing group of each tagging reagent has theformula:

wherein R⁵ and R⁶, independently from one another, are selected from thegroup consisting of hydrogen, deuterium, branched and unbranched C₁ toC₁₂ alkyl groups, C₁ to C₁₂ cycloalkyl groups, C₁ to C₁₂ alkenyl groups,C₁ to C₁₂ cycloalkenyl groups, C₄ to C₁₂ aryl groups and C₄ to C₁₂arylalkyl groups, wherein each of R⁵ and R⁶ optionally contains one ormore ¹³C atoms and one or more deuterium atoms; C^(V) and C^(x),independently of one another, are ¹²C or ¹³C, O^(U) and O^(y),independently of one another, are ¹⁶O or ¹⁸O; and N^(W) is ¹⁴N or ¹⁵N.Preferably, R⁶ is hydrogen or deuterium.

Preferably, R⁵ is selected from the group consisting of hydrogen,deuterium, branched and unbranched C₁ to C₈ alkyl groups, C₁ to C₈cycloalkyl groups, C₁ to C₈ alkenyl groups, C₁ to C₈ cycloalkenylgroups, C₆ to C₁₂ aryl groups and C₆ to C₁₂ arylalkyl groups. In oneembodiment, R⁵ is selected from the group consisting of hydrogen,deuterium, branched and unbranched C₁ to C₄ alkyl groups, C₁ to C₄cycloalkyl groups, and C₂ to C₄ alkenyl groups.

In an embodiment, the balancing group is an “amino acid based balancinggroup”, which means that the balancing group comprises an amino acid,where the carboxyl group forms a bond with the aldehyde, ketone, orcarboxylic acid reactive group or the target molecule, and theN-terminus forms a peptide bond with a second amino acid. The carbonylgroup of the second amino acid will also be part of the balancing group.

In an embodiment, the reporter group of each tagging reagent has theformula:

R², R³ and R⁴, independently of one another, are selected from the groupconsisting of hydrogen, deuterium, branched and unbranched C₁ to C₁₂alkyl groups, C₁ to C₁₂ cycloalkyl groups, C₁ to C₁₂ alkenyl groups, C₁to C₁₂ cycloalkenyl groups, C₄ to C₁₂ aryl groups and C₄ to C₁₂arylalkyl groups, wherein each of R², R³ and R⁴ optionally contains oneor more ¹³C atoms and one or more deuterium atoms; and N^(z) is ¹⁴N or¹⁵N. In a further embodiment, R² and R³, independently of one another,are CH₃, ¹³CH₃, CDH₂, ¹³CDH₂, CD₂H, ¹³CD₂H, CD₃ or ¹³CD₃. In a furtherembodiment, at least one of R² or R³ contains a deuterium atom, andN^(z) is ¹⁵ or N^(W) is ¹⁵N.

Preferably, R⁴ is selected from the group consisting of branched andunbranched C₁ to C₁₂ alkyl groups, C₁ to C₁₂ cycloalkyl groups, C₁ toC₁₂ alkenyl groups, C₁ to C₁₂ cycloalkenyl groups, C₆ to C₁₈ aryl groupsand C₆ to C₁₈ arylalkyl groups. In one embodiment, R⁴ is selected fromthe group consisting of branched and unbranched C₁ to C₃ alkyl groups,C₁ to C₃ cycloalkyl groups, and C₂ to C₃ alkenyl groups. Preferably, atleast one of R² or R³ contains a deuterium atom, N^(z) is ¹⁵N, or N^(W)is ¹⁵N.

In an embodiment, the reporter group is an “N,N-dimethylated amino acidbased reporter group”, which means that the reporter group comprises anN,N-dimethylated amino acid with the exception that the carbonyl groupof the amino acid will not form part of the reporter group.

The aldehyde, ketone, or carboxylic acid reactive group of each taggingreagent can be any functional group able to react with an aldehyde,ketone, or carboxylic acid group of a molecule, such as a glycosylatedpeptide, thereby forming bond between the molecule and the balancinggroup of the tagging reagent. In an embodiment, the aldehyde, ketone, orcarboxylic acid reactive group is an aminooxy group or is —HNNH₂. In anembodiment, the aldehyde, ketone, or carboxylic acid reactive group is ahydrazide, hydrazine, amine, or oxyamine.

In a further embodiment, the dimethylated amino acid based reportergroup is selected from:

In a further embodiment, the amino acid based balancing group isselected from:

Labeling the target molecules comprises the step of reacting thealdehyde, ketone, or carboxylic acid reactive group of the taggingreagents with an aldehyde, ketone, or carboxylic acid group of thetarget molecule.

Methods of Analyzing a Mixture Using 16-Plex Tagging Reagents

The tagging reagents of the present invention also allow samples to belabeled with up to 16 tagging reagents, where each of the taggingreagents have the same aggregate mass but where the mass of eachreporter group and balancing group are different. Where the taggingreagents comprise two amino acids as described above, these 16-plexreagents are easily produced by swapping the amino acid of the balancinggroup with the amino acid of the reporter group. Because the same aminoacids are used in the tagging reagent, the overall mass will remain thesame. However, the mass of the reporter group, and its differentisotopic variations, will be different because the reporter group hasnow been changed. This requires that the two amino acids used in thetagging reagents be different and that the two amino acids havedifferent masses.

For example, an 8-plex series of tagging reagents can compriseN,N-dimethyl leucine (which will make up the reporter group) and alanine(which will make up the balancing group) as depicted in Scheme 3 below.This series of tagging reagents can be expanded to a 16-plex series byusing tagging reagents that have switched the alanine and leucine aminoacids resulting in a reporter group made from the N,N-dimethyl alanineand a balancing group made from leucine. The overall masses of thetagging reagents are the same, but the mass of the different reportergroups will be different for each 16-plex reagent.

Accordingly, one embodiment of the present invention provides a methodof analyzing a mixture containing target molecules comprising the stepsof:

a) labeling target molecules within a first sample with a first taggingreagent, thereby generating first labeled target molecules, wherein thefirst tagging reagent has the formula:

(CH₃)₂-AA¹-AA²-R¹  (formula 3)

where R¹ is an aldehyde, ketone, or carboxylic acid reactive group; AA¹is a first amino acid having an N-terminus; AA² is a second amino acidhaving an N-terminus; and the two CH₃ groups are attached to theN-terminus of AA¹;b) labeling target molecules within one or more additional samples withone or more additional tagging reagents, thereby generating additionallabeled target molecules, wherein at least one of the additional taggingreagents has the formula:

(CH₃)₂-AA²-AA¹-R¹  (formula 4)

where AA¹ and AA² in the additional tagging reagent are the same aminoacids as in the first tagging reagent with the exception that the aminoacids may contain different isotopes; and the two CH₃ groups areattached to the N-terminus of AA² in the additional tagging reagent;c) combining the labeled target molecules of steps a) and b);d) fragmenting the combined labeled target molecules; ande) analyzing the fragments of the labeled molecules.

In a further embodiment, AA¹ and AA², independently from one another,are any natural or synthetic amino acid with the provision that AA¹ andAA² cannot be the same amino acid. AA¹ and AA² also cannot have the samemass. Preferably, the amino acid is a natural amino acid selected fromthe group consisting of leucine, isoleucine, alanine, glycine, valine,histidine, phenylalanine, tryptophan, lysine and tyrosine. Even morepreferably, the natural amino acid is selected from the group consistingof leucine, isoleucine, alanine, glycine and valine. Alternatively, AA¹,AA², or both, are unnatural, non-standard or synthetic amino acidsincluding, but not limited to, β amino acids, norleucine, norvaline,2-aminobutylric acid, 3-aminoisobutylric acid, and 3-aminobutylric acid.

Each tagging reagent comprises a reporter group, an aldehyde, ketone, orcarboxylic acid reactive group, and a balancing group located betweenthe reporter group and aldehyde, ketone, or carboxylic acid reactivegroup, wherein one or more atoms in the reporter group, balancing group,or both, are heavy isotope versions of the atom. The reporter group ofeach tagging reagent has a mass different than the reporter groups ofthe other tagging reagents, the balancing group of each tagging reagenthas a mass different than the balancing groups of other taggingreagents, and the aggregate mass of the reporter group plus thebalancing group for each tagging reagent is the same.

Each tagging reagent is able to generate an immonium ion during thefragmentation step. This method allows for anywhere between 2 to 16samples to be labeled with 2 to 16 tagging reagents. In one embodiment,2 to 8 samples are labeled wherein the generated reporter ions from eachtagging reagent has a mass that differs from the generated reporter ionsfrom the other tagging reagents by two or more Daltons.

In a further embodiment, the balancing group of the 16-plex taggingreagents has the formula:

wherein R⁵ and R⁶, independently from one another, are selected from thegroup consisting of hydrogen, deuterium, branched and unbranched C₁ toC₁₂ alkyl groups, C₁ to C₁₂ cycloalkyl groups, C₁ to C₁₂ alkenyl groups,C₁ to C₁₂ cycloalkenyl groups, C₄ to C₁₂ aryl groups and C₄ to C₁₂arylalkyl groups, wherein each of R⁵ and R⁶ optionally contains one ormore ¹³C atoms and one or more deuterium atoms; C^(V) and C^(x),independently of one another, are ¹²C or ¹³C; O^(U) and O^(y),independently of one another, are ¹⁶O or ¹⁸O; N^(W) is ¹⁴N or ¹⁵N.Preferably, R⁶ is hydrogen or deuterium.

Preferably, R⁵ is selected from the group consisting of hydrogen,deuterium, branched and unbranched C₁ to C₈ alkyl groups, C₁ to C₈cycloalkyl groups, C₁ to C₈ alkenyl groups, C₁ to C₈ cycloalkenylgroups, C₆ to C₁₂ aryl groups and C₆ to C₁₂ arylalkyl groups. In oneembodiment, R⁵ is selected from the group consisting of hydrogen,deuterium, branched and unbranched C₁ to C₄ alkyl groups, C₁ to C₄cycloalkyl groups, and C₂ to C₄ alkenyl groups.

In a further embodiment, the reporter group of the 16-plex taggingreagents has the formula:

In a further embodiment, R² and R³, independently of one another, areCH₃, ¹³CH₃, CDH₂, ¹³CDH₂, CD₂H, ¹³CD₂H, CD₃ or ¹³CD₃. In a furtherembodiment, at least one of R² or R³ contains a deuterium atom, andN^(z) is ¹⁵ or N^(W) is ¹⁵N.

Preferably, R⁴ is selected from the group consisting of branched andunbranched C₁ to C₁₂ alkyl groups, C₁ to C₁₂ cycloalkyl groups, C₁ toC₁₂ alkenyl groups, C₁ to C₁₂ cycloalkenyl groups, C₆ to C₁₈ aryl groupsand C₆ to C₁₈ arylalkyl groups. In one embodiment, R⁴ is selected fromthe group consisting of branched and unbranched C₁ to C₃ alkyl groups,C₁ to C₃ cycloalkyl groups, and C₂ to C₃ alkenyl groups. Preferably, atleast one of R² or R³ contains a deuterium atom, N^(z) is ¹⁵N, or N^(W)is ¹⁵N.

A further embodiment comprises labeling target molecules with from 2 to8 tagging reagents having the formula (CH₃)₂-AA¹-AA²-R¹. A furtherembodiment comprises labeling target molecules with from 2 to 8 taggingreagents having the formula (CH₃)₂-AA²-AA¹-R¹.

In a further embodiment, AA¹ is leucine and AA² is selected from thegroup consisting of alanine, glycine and valine.

In a further embodiment, AA¹ is isoleucine and AA² is selected from thegroup consisting of alanine, glycine and valine.

In a further embodiment, AA¹ is alanine and AA² is selected from thegroup consisting of leucine, isoleucine, glycine and valine.

In a further embodiment, AA¹ is glycine and AA² is selected from thegroup consisting of leucine, isoleucine, alanine and valine.

In a further embodiment, AA¹ is valine and AA² is selected from thegroup consisting of leucine, isoleucine, glycine and alanine.

In some instances, switching the amino acid positions may result indifferent ionization efficiency during fragmentation, different elutiontime, and different reporter ion yields. Accordingly, it may bebeneficial to introduce means for correcting or normalizing thedifferences caused by the switching of the amino acid positions in thetagging reagents. For example, the same amount of a standard could beadded to each sample, and/or the samples could be labeled with adifferent tagging system. A correction factor can then be generated fromMS analysis of these samples by comparing the different signal responseof the same standard compound in different samples.

Tagging Reagent Compounds and Kits

In one embodiment, the present invention provides mass spectrometrytagging reagents comprising a compound having the formula of:

R², R³, R⁴, R⁵ and R⁶, independently of one another, are selected fromthe group consisting of hydrogen, deuterium, branched and unbranched C₁to C₁₂ alkyl groups, C₁ to C₁₂ cycloalkyl groups, C₁ to C₁₂ alkenylgroups, C₁ to C₁₂ cycloalkenyl groups, C₄ to C₁₂ aryl groups and C₄ toC₁₂ arylalkyl groups, wherein each of R², R³, R⁴, R⁵ and R⁶ optionallycontain one or more ¹³C atoms and one or more deuterium atoms; C^(V) andC^(x), independently of one another, are ¹²C or ¹³C; O^(U) and O^(y),independently of one another, are 160 or ¹⁸O; and N^(z) and N^(W),independently of one another, are ¹⁴N or ¹⁵N. In a further embodiment,R² and R³, independently of one another, are CH₃, ¹³CH₃, CDH₂, ¹³CDH₂,CD₂H, ¹³CD₂H, CD₃ or ¹³CD₃. In a further embodiment, R⁶ is hydrogen ordeuterium. Preferably at least one of R² or R³ contains a deuteriumatom, N^(z) is ¹⁵N, or N^(W) is ¹⁵N. Preferably these tagging reagentsare able to generate an immonium ion during fragmentation.

Preferably, R⁴ and R⁵ are selected from the group consisting ofhydrogen, deuterium, branched and unbranched C₁ to C₈ alkyl groups, C₁to C₈ cycloalkyl groups, C₁ to C₈ alkenyl groups, C₁ to C₈ cycloalkenylgroups, C₆ to C₁₂ aryl groups and C₆ to C₁₂ arylalkyl groups. In oneembodiment, R⁴ and R⁵ are selected from the group consisting ofhydrogen, deuterium, branched and unbranched C₁ to C₄ alkyl groups, C₁to C₄ cycloalkyl groups, and C₂ to C₄ alkenyl groups.

In a further embodiment, the tagging reagent comprises a compound havingthe formula:

where R⁵ is selected from the group consisting of: a methyl groupoptionally containing one or more deuterium atoms and wherein the carbonis ¹²C or ¹³C; hydrogen; deuterium; and an isopropyl group optionallycontaining one or more deuterium atoms and one or more ¹³C atoms.

In a further embodiment, the tagging reagent comprises a compound havingthe formula:

where R⁵ is selected from the group consisting of: a methyl groupoptionally containing one or more deuterium atoms and wherein the carbonis ¹²C or ¹³C; hydrogen; deuterium; and a butyl group optionallycontaining one or more deuterium atoms and one or more ¹³C atoms.

In a further embodiment, the tagging reagent comprises a compound havingthe formula:

where R⁵ is selected from the group consisting of: a methyl groupoptionally containing one or more deuterium atoms and wherein the carbonis ¹²C or ¹³C; an isopropyl group optionally containing one or moredeuterium atoms and one or more ¹³C atoms; and a butyl group optionallycontaining one or more deuterium atoms and one or more ¹³C atoms.

In a further embodiment, the tagging reagent comprises a compound havingthe formula:

where R⁵ is selected from the group consisting of: hydrogen; deuterium;an isopropyl group optionally containing one or more deuterium atoms andone or more ¹³C atoms; and a butyl group optionally containing one ormore deuterium atoms and one or more ¹³C atoms.

In a further embodiment, the tagging reagent comprises a compound havingthe formula:

where R⁵ is selected from the group consisting of: a methyl groupoptionally containing one or more deuterium atoms and wherein the carbonis ¹²C or ¹³C; hydrogen; deuterium; and an isopropyl group optionallycontaining one or more deuterium atoms and one or more ¹³C atoms.

The present invention also provides a kit comprising two or more taggingreagents, wherein each tagging reagent comprises a reporter group, analdehyde, ketone, or carboxylic acid reactive group, and a balancinggroup located between the reporter group and aldehyde, ketone, orcarboxylic acid reactive group, wherein one or more atoms in thereporter group, balancing group, or both, are isotopically heavyversions of the atom; and wherein the reporter group of each taggingreagent has a mass different than the reporter groups of the othertagging reagents, the balancing group of each tagging reagent has a massdifferent than the balancing groups of other tagging reagents, and theaggregate mass of the reporter group plus the balancing group for eachtagging reagent is the same. Preferably, the tagging reagents are ableto generate an immonium ion.

In an embodiment, the balancing group of each tagging reagent has theformula:

wherein R⁵ and R⁶, independently from one another, are selected from thegroup consisting of hydrogen, deuterium, branched and unbranched C₁ toC₁₂ alkyl groups, C₁ to C₁₂ cycloalkyl groups, C₁ to C₁₂ alkenyl groups,C₁ to C₁₂ cycloalkenyl groups, C₄ to C₁₂ aryl groups and C₄ to C₁₂arylalkyl groups, wherein each of R⁵ and R⁶ optionally contains one ormore ¹³C atoms and one or more deuterium atoms; C^(V) and C^(x),independently of one another, are ¹²C or ¹³C, O^(U) and O^(y),independently of one another, are ¹⁶O or ¹⁸O; and N^(W) is ¹⁴N or ¹⁵N.In a further embodiment, R⁶ is hydrogen or deuterium.

Preferably, R⁵ is selected from the group consisting of hydrogen,deuterium, branched and unbranched C₁ to C₈ alkyl groups, C₁ to C₈cycloalkyl groups, C₁ to C₈ alkenyl groups, C₁ to C₈ cycloalkenylgroups, C₆ to C₁₂ aryl groups and C₆ to C₁₂ arylalkyl groups. In oneembodiment, R⁵ is selected from the group consisting of hydrogen,deuterium, branched and unbranched C₁ to C₄ alkyl groups, C₁ to C₄cycloalkyl groups, and C₂ to C₄ alkenyl groups.

The reporter group of each tagging reagent has the formula:

wherein R², R³ and R⁴, independently of one another, are selected fromthe group consisting of hydrogen, deuterium, branched and unbranched C₁to C₁₂ alkyl groups, C₁ to C₁₂ cycloalkyl groups, C₁ to C₁₂ alkenylgroups, C₁ to C₁₂ cycloalkenyl groups, C₄ to C₁₂ aryl groups and C₄ toC₁₂ arylalkyl groups, wherein each of R², R³ and R⁴ optionally containsone or more ¹³C atoms and one or more deuterium atoms; and N^(z) is ¹⁴Nor ¹⁵N. In a further embodiment, R² and R³, independently of oneanother, are CH₃, ¹³CH₃, CDH₂, ¹³CDH₂, CD₂H, ¹³CD₂H, CD₃ or ¹³CD₃. In afurther embodiment, at least one of R² or R³ contains a deuterium atom,N^(z) is ¹⁵N, or N^(W) is ¹⁵N.

Preferably, R⁴ is selected from the group consisting of branched andunbranched C₁ to C₁₂ alkyl groups, C₁ to C₁₂ cycloalkyl groups, C₁ toC₁₂ alkenyl groups, C₁ to C₁₂ cycloalkenyl groups, C₆ to C₁₈ aryl groupsand C₆ to C₁₈ arylalkyl groups. In one embodiment, R⁴ is selected fromthe group consisting of branched and unbranched C₁ to C₃ alkyl groups,C₁ to C₃ cycloalkyl groups, and C₂ to C₃ alkenyl groups. Preferably, atleast one of R² or R³ contains a deuterium atom, N^(z) is ¹⁵N, or N^(W)is ¹⁵N.

The aldehyde, ketone, or carboxylic acid reactive group of each taggingreagent can be any functional group able to react with an amine group ofa peptide or small molecule, thereby forming bond between the peptideand the balancing group of the tagging reagent. In one embodiment, thealdehyde, ketone, or carboxylic acid reactive group is an aminooxy groupor is —HNNH₂.

In one embodiment, the reporter groups of the tagging reagents of thepresent invention are derived from natural amino acids where the aminogroup in the amino acid which makes up the reporter group has beendimethylated.

The present invention also provides a kit where at least one taggingreagent has the formula:

(CH₃)₂-AA¹-AA²-R¹  (formula 3)

where R¹ is an aldehyde, ketone, or carboxylic acid reactive group; AA¹is a first amino acid having an N-terminus; AA² is a second amino acidhaving an N-terminus; and the two CH₃ groups are attached to theN-terminus of AA¹; and

wherein at least one tagging reagent has the formula:

(CH₃)₂-AA²-AA¹-R¹  (formula 4)

where AA¹ and AA² are the same amino acids as in the first taggingreagent with the exception that the amino acids may contain differentisotopes; and the two CH₃ groups are attached to the N-terminus of AA²;

AA¹ and AA², independently from one another, are any amino acid,preferably a natural amino acid, with the provision that AA¹ and AA²cannot be the same amino acid or have the same mass. Preferably, AA¹ andAA², independently from one another, are selected from the groupconsisting of leucine, isoleucine, alanine, glycine and valine.

In a further embodiment, the kit comprises 2 to 8 tagging reagentshaving the formula (CH₃)₂-AA¹-AA²-R¹. In another embodiment, the kitcomprises 2 to 8 tagging reagents having the formula (CH₃)₂-AA²-AA¹-R¹.

In one embodiment, the kit comprises two or more tagging reagents, threeor more tagging reagents, four or more tagging reagents, five or moretagging reagents, six or more tagging reagents, seven or more taggingreagents, eight or more tagging reagents, nine or more tagging reagents,ten or more tagging reagents, eleven or more tagging reagents, twelve ormore tagging reagents, thirteen or more tagging reagents, fourteen ormore tagging reagents, fifteen or more tagging reagents, or sixteen ormore tagging reagents.

The tagging reagents disclosed herein serve as attractive alternativesfor isobaric tag for relative and absolute quantitation (iTRAQ) andtandem mass tags (TMTs) due to their synthetic simplicity, labelingefficiency and improved fragmentation efficiency. Additionally, thesetagging reagents are able to react with aldehyde, ketone, or carboxylicacid groups, allowing them to chemically tag glycans. The taggingreagents disclosed herein enable simultaneous quantitation of multipleglycan/glycan samples and identification based on sequence-specificfragmentation. The isobaric reagents can be synthesized in fewer stepsusing commercially available reagents (one step or two step synthesis),thus offering synthetic simplicity and much reduced cost as compared toother existing technology. This feature allows the routine applicationof these isobaric tagging reagents to many large-scale proteomic andglycomic studies. In addition to multiplexed quantitation, the reagentsdisclosed herein based on dimethylated amino acid tagging promoteenhanced fragmentation, thus enabling more confident proteinidentification and superior capability for de novo sequencing. Thisfeature makes the present invention an important tool for identifyingand analyzing glycans in biological studies.

In an embodiment, the invention provides an isotopically enriched samplecomprising any of the compounds disclosed herein, including thedisclosed compounds having specific isotopic compositions, and methodsof using an isotopically enriched sample comprising any of the compoundsdisclosed herein, including the disclosed compounds having specificisotopic compositions. In a specific embodiment, the invention providesan isotopically enriched sample comprising a compound of the inventionhaving a specific isotopic composition, wherein the compound is presentin an abundance that is at least 10 times greater, for some embodimentsat least 100 times greater, for some embodiments at least 1,000 timesgreater, for some embodiments at least 10,000 times greater, than theabundance of the same compound having the same isotopic composition in anaturally occurring sample, and related methods of using these samples,for example for use as a tagging reagent in mass spectrometry. In aspecific embodiment, the invention provides an isotopically enrichedsample having a purity with respect to a compound of the inventionhaving a specific isotopic composition that is substantially enriched,for example, a purity equal to or greater than 90%, in some embodimentsequal to or greater than 95%, in some embodiments equal to or greaterthan 99%, in some embodiments equal to or greater than 99.9%, in someembodiments equal to or greater than 99.99%, and in some embodimentsequal to or greater than 99.999%, and related methods of using thesesamples, for example for use as a tagging reagent in mass spectrometry.In a specific embodiment, the invention provides an isotopicallyenriched sample that has been purified with respect to a compound of theinvention having a specific isotopic composition, for example usingisotope purification methods known in the art, and related methods ofusing these samples, for example for use as a tagging reagent in massspectrometry.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates different types of glycosylation of peptides,including O-glycans and N-glycans.

FIG. 2 illustrates a carboxylic acid labeling scheme for peptides andfatty acids according to an embodiment of the present invention.

FIG. 3 shows exemplary fluorescent agents used to detect the presence ofglycans.

FIG. 4 shows isotopically labeled AminoxyTMT reagents.

FIG. 5 illustrates limitations of AminoxyTMT reagents including MSspectra obtained using AminoxyTMT reagents at low collisional energy andhigh collisional energy.

FIG. 6 shows QUANTITY isobaric tags used for glycan detection includingproposed fragmentation pathways.

FIG. 7 shows exemplary aldehyde, ketone, or carboxylic acid reactivegroups for SUGAR tags in an embodiment of the invention.

FIG. 8 shows DiLeu-based labeling tags (A-DiLeu, H-DiLeu, and HG-DiLeu)for glycan detection (top), and a synthesis pathway for producing theA-DiLeu glycan tag (bottom) according to an embodiment of the presentinvention.

FIG. 9 shows a synthesis pathway for producing the H-DiLeu glycan tagaccording to an embodiment of the present invention.

FIG. 10 shows a synthesis pathway or producing the HG-DiLeu glycan tagaccording to an embodiment of the present invention.

FIG. 11 shows isotope positions and synthesis of a 4-plex HG-DiLeu(SUGAR) tagging system in an embodiment of the invention.

FIG. 12 shows isotope positions of a 12-plex HG-DiLeu (SUGAR) taggingsystem in an embodiment of the invention.

FIG. 13 shows a MS¹ spectrum of SUGAR labeling with (Glc)₈ as a glycanstandard in an embodiment of the present invention. In this example,over 85% yield was achieved by SUGAR labeling.

FIG. 14 shows a MS² fragmentation spectrum of a SUGAR labeled (Glc)₈.All the Y ions could be identified with high intensity of the reporterion (shown at the peak labeled with star) in this example.

FIGS. 15 and 16 show MS² fragmentation spectra illustratingquantification accuracy of labeled (Glc)₈. Either 1:1:1:1 or 10:1 ratiosprovide accurate result within a 15% relative error.

FIG. 17 shows labeling comparison of an AminoxyTMT tag and a SUGAR tag.FIG. 17 , panel a, shows an unlabeled glycan released from aglycoprotein standard, bovine thyroglobulin (BTG). FIG. 17 , panel b,shows partial labeling after reaction with AminoxyTMT. FIG. 17 , panelc, shows complete labeling after reaction with the SUGAR tag.

FIG. 18 illustrates the labeling efficiency of the SUGAR tag of FIG. 17with various different glycans. All types of glycans show nearlycomplete labeling to facilitate quantitative glycomics

FIGS. 19 and 20 show the relative abundance of fragments using the SUGARtag of FIG. 17 and AminoxyTMT at different collision energies. SUGAR taglabeled glycans, for both high-mannose and complex type, are able toproduce more backbone fragments as well as higher intensity reporterions.

FIG. 21 shows the quantitation accuracy of a 4-plex SUGAR tagging systemwith various different glycans. The result shows the performance of theSUGAR tagging system with accurate quantification and small deviation.

FIGS. 22-23 show charge switch labeling of a fatty acid with a SUGARtagging system. The free fatty acid could not be identified in positivemode (FIG. 22 , panel a), while the SUGAR labeled fatty acid was able tobe identified (FIG. 22 , panel b). Also, the SUGAR labeled fatty acidwas able to produce a suitable reporter ion during fragmentation tofacilitate quantitative lipidomics.

FIGS. 24-25 show dimethylated peptide mixtures (FIG. 24 ) and the samedimethylated peptide mixtures labeled with a SUGAR tag (FIG. 25 ).

FIG. 26 shows tandem MS fragmentation of SUGAR labeled peptide withextensive backbone b-/y-fragment ions that enabled sequenceidentification. In this instance the peptide issANLmAGHWVAISGAAGGLGSLAVQYAk, where the lower case letters representmodifications at the different position: N-term (Dimethyl), M5(Oxidation), K28 (Dimethyl), C-term (SUGAR-114 tag).

FIG. 27 shows a workflow for quantitative analysis of a glycosylatedprotein (e.g., bovine thyroglobulin) with a 4-plex HG-DiLeu (SUGAR)tagging system in an embodiment of the present invention. The protein isdenatured and the glycans released from the peptide using PNGase F. Theglycans are then reacted with the tagging reagents followed by MSanalysis.

FIG. 28 shows the structure and isotope configurations of exemplary4-plex SUGAR tags. Black dots: ¹³C, grey dots: ²H, white dots: ¹⁵N.

FIG. 29 shows relative quantification performance of 4-plexSUGAR-labeled N-glycans released from BTG. Labeled N-glycans were mixedat ratios of 1:1:5:10 and analyzed in triplicate. The reporter ionintensity ratio results of 116/115, 117/115 and 118/115 are plotted atthe log scale. Box plots show the median (line), the 25^(th) and 75^(th)percentile (box), and the 5^(th) and 95^(th) percentile (whiskers)(panel A). Representative MS spectra reporter ion range forSUGAR-labeled N-glycans H₄N₃FS (panel B) and H₆N₄FS (panel C) are shown.

FIG. 30 shows selected N-glycan relative quantification of equal amountsof human serum protein from ALL patients before (SUGAR-115), 1 month(SUGAR-116), 3 months (SUGAR-117), and 6 months (SUGAR-118) afterinduction chemotherapy. Ratios represent intensities of reporter ionsfor SUGAR-labeled N-glycans. Error bars represent the standard deviationof three biological replicates.

FIG. 31 illustrates exemplary isotope configurations of 12-plex SUGARtags. Black dots: ¹³C, grey dots: ²H, white dots: ¹⁵N.

FIG. 32 shows MALDI-MS spectra of one step labeling and stepwiselabeling. With stepwise labeling, higher labeling efficiency wasobserved in half the labeling time with minimal glycan reduction.

FIG. 33 illustrates representative MS spectra reporter ion ranges forSUGAR-labeled H₅N₄F₃S and H₅N₃S at 1:1:1:1 (top panels) and 10:5:1:1ratios (bottom panels).

FIG. 34 shows ¹H NMR of an exemplary SUGAR tag.

FIG. 35 shows ¹³C NMR of an exemplary SUGAR tag.

FIG. 36 shows HCD MS/MS fragmentation of a SUGAR-labeled N-glycan H₄N₃Sat NCE 30 (panel A), dimethylated peptide at NCE 30 (panel B), and oleicacid at NCE 25 (panel C). The star is a reporter ion upon fragmentation.

FIGS. 37 and 38 show MS spectra of non-labeled and SUGAR labeledsteroids, 3β-hydroxypregn-5-en-20-one (FIG. 37 ) and4-androsten-11β,17α-diol-3-one-17β-carboxylic acid (FIG. 38 ).

FIG. 39 shows exemplary mass defect SUGAR reagents for MS¹identification and quantification of glycans. Each reagent has a uniquecombination of heavy isotope substitutions resulting in a massdifference of less than 25 mDa between a reagent and the next closestreagent.

FIG. 40 show mass spectra of different compounds tagged with the massdefect SUGAR reagents of FIG. 39 . The tagged compounds were provided inan approximate 1:5:10 ratio which approximately corresponds to therelative abundances shown in the mass spectra.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein the terms “tagging” and “labeling” refers to reacting areagent or compound with a molecule of interest, including but notlimited to glycans and glycosylated peptides, so that one or morefunctional groups are attached to the molecule of interest. A “tagged”or “labeled” molecule of interest refers to a molecule of interesthaving the one or more functional groups attached.

“Glycosylation” refers to the process in which a carbohydrate isattached to a functional group of another molecule, such as a peptide.In particular, glycosylation includes the enzymatic process thatattaches glycans to proteins, lipids, or other organic molecules.Examples of glycosylation includes the following:

-   -   N-linked glycans attached to a nitrogen of asparagine or        arginine side-chains. N-linked glycosylation requires        participation of a special lipid called dolichol phosphate;    -   O-linked glycans attached to the hydroxyl oxygen of serine,        threonine, tyrosine, hydroxylysine, or hydroxyproline        side-chains, or to oxygen atoms on lipids such as ceramide;    -   phospho-glycans linked through the phosphate of a        phospho-serine;    -   C-linked glycans, a rare form of glycosylation where a sugar is        added to a carbon on a tryptophan side-chain; and    -   glypiation, which is the addition of a GPI anchor that links        proteins to lipids through glycan linkages.

The term “glycan” is used herein interchangeably with the terms“polysaccharide” and “carbohydrate” and refers to any biologicallyoccurring form (N- or O-linked glycans, glycolipids, glycosaminoglycans,microbial polysaccharides) each having its own chemical repertoire ofpresentation or modifications.

The terms “peptide” and “polypeptide” are used synonymously in thepresent disclosure, and refer to a class of compounds composed of aminoacid residues chemically bonded together by amide bonds (or peptidebonds). Peptides are polymeric compounds comprising at least two aminoacid residues or modified amino acid residues. Peptides includecompositions comprising a few amino acids and include compositionscomprising intact proteins or modified proteins. Modifications can benaturally occurring or non-naturally occurring, such as modificationsgenerated by chemical synthesis. Modifications to amino acids inpolypeptides include, but are not limited to, phosphorylation,glycosylation, lipidation, prenylation, sulfonation, hydroxylation,acetylation, methionine oxidation, alkylation, acylation, carbamylation,iodination and the addition of cofactors. Peptides include proteins andfurther include compositions generated by degradation of proteins, forexample by proteolytic digestion. Peptides and polypeptides may begenerated by substantially complete digestion or by partial digestion ofproteins. Identifying or sequencing a peptide refers to determination ofis composition, particularly its amino acid sequence, andcharacterization of any modifications of one or more amino acidscomprising the peptide or polypeptide.

“Fragment” refers to a portion of molecule, such as a glycosylatedpeptide. Fragments may be singly or multiply charged ions. Fragments maybe derived from bond cleavage in a parent molecule, including sitespecific cleavage of polypeptide bonds in a parent peptide. Fragmentsmay also be generated from multiple cleavage events or steps. Fragmentsmay be a truncated peptide, either carboxy-terminal, amino-terminal orboth, of a parent peptide. A fragment may refer to products generatedupon the cleavage of a polypeptide bond, a C—C bond, a C—N bond, a C—Obond or combination of these processes. Fragments may refer to productsformed by processes whereby one or more side chains of amino acids areremoved, or a modification is removed, or any combination of theseprocesses. Fragments useful in the present invention include fragmentsformed under metastable conditions or result from the introduction ofenergy to the precursor by a variety of methods including, but notlimited to, collision induced dissociation (CID), higher-energycollision dissociation (HCD), surface induced dissociation (SID), laserinduced dissociation (LID), electron capture dissociation (ECD),electron transfer dissociation (ETD), ultraviolet photo-dissociation(UVPD), or any combination of these methods or any equivalents known inthe art of tandem mass spectrometry. Fragments useful in the presentinvention also include, but are not limited to, x-type fragments, y-typefragments, z-type fragments, a-type fragments, b-type fragments, c-typefragments, internal ion (or internal cleavage ions), immonium ions orsatellite ions. The types of fragments derived from a parent analyte,such as a glycosylated polypeptide analyte, often depend on the sequenceof the parent, method of fragmentation, charge state of the parentprecursor ion, amount of energy introduced to the parent precursor ionand method of delivering energy into the parent precursor ion.Properties of fragments, such as molecular mass, may be characterized byanalysis of a fragmentation mass spectrum.

An “aldehyde, ketone, or carboxylic acid reactive group” of a taggingreagent can be any functional group able to react with an amine group ofa peptide or small molecule, thereby forming bond between the peptide orsmall molecule and the balancing group of the tagging reagent. An“aldehyde” generally refers to an organic compound having a functionalgroup with the structure —CHO. A “ketone” generally refers to an organiccompound having a functional group with the structure RC(═O)R′, where Rand R′ can be a variety of carbon-containing substituents. A “carboxylicacid” generally refers to an organic compound having a functional groupwith the structure R-GOGH.

An “amino acid” refers to an organic compound containing an amino group(NH₂), a carboxylic acid group (COOH), and any of various organic sidegroups that have the basic formula NH₂CHRCOOH. Natural amino acids arethose amino acids which are produced in nature, such as isoleucine,alanine, leucine, asparagine, lysine, aspartic acid, methionine,cysteine, phenylalanine, glutamic acid, threonine, glutamine,tryptophan, glycine, valine, proline, serine, tyrosine, arginine, andhistidine as well as ornithine and selenocysteine.

The term “alkyl” refers to a monoradical of a branched or unbranched(straight-chain or linear) saturated hydrocarbon and to cycloalkylgroups having one or more rings. Alkyl groups as used herein includethose having from 1 to 30 carbon atoms, preferably having from 1 to 12carbon atoms. Alkyl groups include small alkyl groups having 1 to 3carbon atoms. Alkyl groups include medium length alkyl groups havingfrom 4-10 carbon atoms. Alkyl groups include long alkyl groups havingmore than 10 carbon atoms, particularly those having 10-30 carbon atoms.Cycloalkyl groups include those having one or more rings. Cyclic alkylgroups include those having a 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11- or12-member carbon ring and particularly those having a 3-, 4-, 5-, 6-, or7-member ring. The carbon rings in cyclic alkyl groups can also carryalkyl groups. Cyclic alkyl groups can include bicyclic and tricyclicalkyl groups. Alkyl groups are optionally substituted. Substituted alkylgroups include among others those which are substituted with arylgroups, which in turn can be optionally substituted. Specific alkylgroups include methyl, ethyl, n-propyl, iso-propyl, cyclopropyl,n-butyl, s-butyl, t-butyl, cyclobutyl, n-pentyl, branched-pentyl,cyclopentyl, n-hexyl, branched hexyl, and cyclohexyl groups, all ofwhich are optionally substituted. Substituted alkyl groups include fullyhalogenated or semihalogenated alkyl groups, such as alkyl groups havingone or more hydrogens replaced with one or more fluorine atoms, chlorineatoms, bromine atoms and/or iodine atoms. Substituted alkyl groupsinclude fully fluorinated or semifluorinated alkyl groups, such as alkylgroups having one or more hydrogens replaced with one or more fluorineatoms. An alkoxy group is an alkyl group linked to oxygen and can berepresented by the formula R—O. Examples of alkoxy groups include, butare not limited to, methoxy, ethoxy, propoxy, butoxy and heptoxy. Alkoxygroups include substituted alkoxy groups wherein the alky portion of thegroups is substituted as provided herein in connection with thedescription of alkyl groups.

The term “alkenyl” refers to a monoradical of a branched or unbranchedunsaturated hydrocarbon group having one or more double bonds and tocycloalkenyl groups having one or more rings wherein at least one ringcontains a double bond. Alkenyl groups include those having 1, 2 or moredouble bonds and those in which two or more of the double bonds areconjugated double bonds. Alkenyl groups include those having from 1 to20 carbon atoms, preferably having from 1 to 12 carbon atoms. Alkenylgroups include small alkenyl groups having 2 to 3 carbon atoms. Alkenylgroups include medium length alkenyl groups having from 4-10 carbonatoms. Alkenyl groups include long alkenyl groups having more than 10carbon atoms, particularly those having 10-20 carbon atoms. Cycloalkenylgroups include those having one or more rings. Cyclic alkenyl groupsinclude those in which a double bond is in the ring or in an alkenylgroup attached to a ring. Cyclic alkenyl groups include those having a3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11- or 12-member carbon ring andparticularly those having a 3-, 4-, 5-, 6- or 7-member ring. The carbonrings in cyclic alkenyl groups can also carry alkyl groups. Cyclicalkenyl groups can include bicyclic and tricyclic alkyl groups. Alkenylgroups are optionally substituted. Substituted alkenyl groups includeamong others those which are substituted with alkyl or aryl groups,which groups in turn can be optionally substituted. Specific alkenylgroups include ethenyl, prop-1-enyl, prop-2-enyl, cycloprop-1-enyl,but-1-enyl, but-2-enyl, cyclobut-1-enyl, cyclobut-2-enyl, pent-1-enyl,pent-2-enyl, branched pentenyl, cyclopent-1-enyl, hex-1-enyl, branchedhexenyl, cyclohexenyl, all of which are optionally substituted.Substituted alkenyl groups include fully halogenated or semihalogenatedalkenyl groups, such as alkenyl groups having one or more hydrogensreplaced with one or more fluorine atoms, chlorine atoms, bromine atomsand/or iodine atoms. Substituted alkenyl groups include fullyfluorinated or semifluorinated alkenyl groups, such as alkenyl groupshaving one or more hydrogens replaced with one or more fluorine atoms.

The term “aryl” refers to a chemical group having one or more 5-, 6- or7-member aromatic or heterocyclic aromatic rings. An aromatichydrocarbon is a hydrocarbon with a conjugated cyclic molecularstructure. Aryl groups include those having from 4 to 30 carbon atoms,preferably having from 6 to 18 carbon atoms. Aryl groups can contain asingle ring (e.g., phenyl), one or more rings (e.g., biphenyl) ormultiple condensed (fused) rings, wherein at least one ring is aromatic(e.g., naphthyl, dihydrophenanthrenyl, fluorenyl, or anthryl).Heterocyclic aromatic rings can include one or more N, O, or S atoms inthe ring. Heterocyclic aromatic rings can include those with one, two orthree N, those with one or two 0, and those with one or two S, orcombinations of one or two or three N, O or S. Aryl groups areoptionally substituted. Substituted aryl groups include among othersthose which are substituted with alkyl or alkenyl groups, which groupsin turn can be optionally substituted. Specific aryl groups includephenyl groups, biphenyl groups, pyridinyl groups, and naphthyl groups,all of which are optionally substituted. Substituted aryl groups includefully halogenated or semihalogenated aryl groups, such as aryl groupshaving one or more hydrogens replaced with one or more fluorine atoms,chlorine atoms, bromine atoms and/or iodine atoms. Substituted arylgroups include fully fluorinated or semifluorinated aryl groups, such asaryl groups having one or more hydrogens replaced with one or morefluorine atoms. Aryl groups include, but are not limited to, aromaticgroup-containing or heterocylic aromatic group-containing groupscorresponding to any one of the following benzene, naphthalene,naphthoquinone, diphenylmethane, fluorene, fluoranthene, anthracene,anthraquinone, phenanthrene, tetracene, naphthacenedione, pyridine,quinoline, isoquinoline, indoles, isoindole, pyrrole, imidazole,oxazole, thiazole, pyrazole, pyrazine, pyrimidine, purine,benzimidazole, furans, benzofuran, dibenzofuran, carbazole, acridine,acridone, phenanthridine, thiophene, benzothiophene, dibenzothiophene,xanthene, xanthone, flavone, coumarin, azulene or anthracycline. As usedherein, a group corresponding to the groups listed above expresslyincludes an aromatic or heterocyclic aromatic radical, includingmonovalent, divalent and polyvalent radicals, of the aromatic andheterocyclic aromatic groups listed above provided in a covalentlybonded configuration in the compounds of the present invention. Arylgroups optionally have one or more aromatic rings or heterocyclicaromatic rings having one or more electron donating groups, electronwithdrawing groups and/or targeting ligands provided as substituents.

Arylalkyl groups are alkyl groups substituted with one or more arylgroups wherein the alkyl groups optionally carry additional substituentsand the aryl groups are optionally substituted. Specific alkylarylgroups are phenyl-substituted alkyl groups, e.g., phenylmethyl groups.Alkylaryl groups are alternatively described as aryl groups substitutedwith one or more alkyl groups wherein the alkyl groups optionally carryadditional substituents and the aryl groups are optionally substituted.Specific alkylaryl groups are alkyl-substituted phenyl groups such asmethylphenyl. Substituted arylalkyl groups include fully halogenated orsemihalogenated arylalkyl groups, such as arylalkyl groups having one ormore alkyl and/or aryl having one or more hydrogens replaced with one ormore fluorine atoms, chlorine atoms, bromine atoms and/or iodine atoms.

Optional substitution of any alkyl, alkenyl and aryl groups includessubstitution with one or more of the following substituents: halogens,—CN, —COOR, —OR, —COR, —OCOOR, —CON(R)₂, —OCON(R)₂, —N(R)₂, —NO₂, —SR,—SO₂R, —SO₂N(R)₂ or —SOR groups. Optional substitution of alkyl groupsincludes substitution with one or more alkenyl groups, aryl groups orboth, wherein the alkenyl groups or aryl groups are optionallysubstituted. Optional substitution of alkenyl groups includessubstitution with one or more alkyl groups, aryl groups, or both,wherein the alkyl groups or aryl groups are optionally substituted.Optional substitution of aryl groups includes substitution of the arylring with one or more alkyl groups, alkenyl groups, or both, wherein thealkyl groups or alkenyl groups are optionally substituted.

Optional substituents for alkyl, alkenyl and aryl groups include amongothers:

—COOR where R is a hydrogen or an alkyl group or an aryl group and morespecifically where R is methyl, ethyl, propyl, butyl, or phenyl groupsall of which are optionally substituted;

—COR where R is a hydrogen, or an alkyl group or an aryl groups and morespecifically where R is methyl, ethyl, propyl, butyl, or phenyl groupsall of which groups are optionally substituted;

—CON(R)₂ where each R, independently of each other R, is a hydrogen oran alkyl group or an aryl group and more specifically where R is methyl,ethyl, propyl, butyl, or phenyl groups all of which groups areoptionally substituted; R and R can form a ring which may contain one ormore double bonds;

—OCON(R)₂ where each R, independently of each other R, is a hydrogen oran alkyl group or an aryl group and more specifically where R is methyl,ethyl, propyl, butyl, or phenyl groups all of which groups areoptionally substituted; R and R can form a ring which may contain one ormore double bonds;

—N(R)₂ where each R, independently of each other R, is an alkyl group,acyl group or an aryl group and more specifically where R is methyl,ethyl, propyl, butyl, or phenyl or acetyl groups all of which areoptionally substituted; or R and R can form a ring which may contain oneor more double bonds.

—SR, —SO₂R, or —SOR where R is an alkyl group or an aryl groups and morespecifically where R is methyl, ethyl, propyl, butyl, phenyl groups allof which are optionally substituted; for —SR, R can be hydrogen;

—OCOOR where R is an alkyl group or an aryl groups;

—SO₂N(R)₂ where R is a hydrogen, an alkyl group, or an aryl group and Rand R can form a ring;

—OR where R is H, alkyl, aryl, or acyl; for example, R can be an acylyielding —OCOR* where R* is a hydrogen or an alkyl group or an arylgroup and more specifically where R* is methyl, ethyl, propyl, butyl, orphenyl groups all of which groups are optionally substituted.

As used herein, the term “alkylene” refers to a divalent radical derivedfrom an alkyl group or as defined herein. Alkylene groups in someembodiments function as attaching and/or spacer groups in the presentcompositions. Compounds of the present invention include substituted andunsubstituted C₁-C₃₀ alkylene, C₁-C₁₂ alkylene and C₁-C₅ alkylenegroups. The term “alkylene” includes cycloalkylene and non-cyclicalkylene groups.

As used herein, the term “cycloalkylene” refers to a divalent radicalderived from a cycloalkyl group as defined herein. Cycloalkylene groupsin some embodiments function as attaching and/or spacer groups in thepresent compositions. Compounds of the present invention includesubstituted and unsubstituted C₁-C₃₀ cycloalkenylene, C₁-C₁₂cycloalkenylene and C₁-C₅ cycloalkenylene groups.

As used herein, the term “alkenylene” refers to a divalent radicalderived from an alkenyl group as defined herein. Alkenylene groups insome embodiments function as attaching and/or spacer groups in thepresent compositions. Compounds of the present invention includesubstituted and unsubstituted C₁-C₂₀ alkenylene, C₁-C₁₂ alkenylene andC₁-C₅ alkenylene groups. The term “alkenylene” includes cycloalkenyleneand non-cyclic alkenylene groups.

As used herein, the term “cycloalkenylene” refers to a divalent radicalderived from a cylcoalkenyl group as defined herein. Cycloalkenylenegroups in some embodiments function as attaching and/or spacer groups inthe present compositions.

Specific substituted alkyl groups include haloalkyl groups, particularlytrihalomethyl groups and specifically trifluoromethyl groups. Specificsubstituted aryl groups include mono-, di-, tri, tetra- andpentahalo-substituted phenyl groups; mono-, di-, tri-, tetra-, penta-,hexa-, and hepta-halo-substituted naphthalene groups; 3- or4-halo-substituted phenyl groups, 3- or 4-alkyl-substituted phenylgroups, 3- or 4-alkoxy-substituted phenyl groups, 3- or4-RCO-substituted phenyl, 5- or 6-halo-substituted naphthalene groups.More specifically, substituted aryl groups include acetylphenyl groups,particularly 4-acetylphenyl groups; fluorophenyl groups, particularly3-fluorophenyl and 4-fluorophenyl groups; chlorophenyl groups,particularly 3-chlorophenyl and 4-chlorophenyl groups; methylphenylgroups, particularly 4-methylphenyl groups, and methoxyphenyl groups,particularly 4-methoxyphenyl groups.

As used herein, the term “halo” refers to a halogen group such as afluoro (—F), chloro (—Cl), bromo (—Br) or iodo (—I).

As to any of the above groups which contain one or more substituents, itis understood, that such groups do not contain any substitution orsubstitution patterns which are sterically impractical and/orsynthetically non-feasible. In addition, the compounds of this inventioninclude all stereochemical isomers arising from the substitution ofthese compounds.

As used herein, “isotopically labeled”, “isotopic”, “isotopes”,“isotope”, “isotopically-different”, “isotopically enriched” and thelike refer to compounds (e.g., tagging reagents, target analytes,labeled samples and end-products, etc.) whereby a process has introducedone or more isotopes into the relevant compound in excess of the naturalisotopic abundance. “Isotopically-heavy” refers to a compound orfragments/moieties thereof that have been enriched with one or more highmass, or heavy isotopes (e.g., stable isotopes such as deuterium, ¹³C,¹⁵N, and ¹⁸O).

In an embodiment, an isotopically enriched sample comprises a compoundof the invention having a specific isotopic composition, wherein thecompound is present in an abundance that is at least 10 times greater,for some embodiments at least 100 times greater, for some embodiments atleast 1,000 times greater, for some embodiments at least 10,000 timesgreater, than the abundance of the same compound having the sameisotopic composition in a naturally occurring sample. In anotherembodiment, an isotopically enriched sample has a purity with respect toa compound of the invention having a specific isotopic composition thatis substantially enriched, for example, a purity equal to or greaterthan 90%, in some embodiments equal to or greater than 95%, in someembodiments equal to or greater than 99%, in some embodiments equal toor greater than 99.9%, in some embodiments equal to or greater than99.99%, and in some embodiments equal to or greater than 99.999%. Inanother embodiment, an isotopically enriched sample is a sample that hasbeen purified with respect to a compound of the invention having aspecific isotopic composition, for example using isotope purificationmethods known in the art.

Isobaric Labeling

Numerous MS-based chemical derivatization quantitation approaches havebeen developed and widely used for quantitative proteomics andpeptidomics (Ong et al., Nat. Chem. Biol. 2005, 1:252-262).Mass-difference labeling approaches introduce a mass difference for thesame peptide by incorporating a light or heavy isotopic form of thelabeling reagent. Light and heavy labeled peptides are combined prior toMS analysis, and quantitation is accomplished by comparing the extractedion chromatogram peak areas of light and heavy forms of the samepeptide. Methods such as isotope-coded affinity tags (ICAT), stableisotope labeling with amino acids in cell culture (SILAC),4-trimethylammonium-butyryl (TMAB) labels, and reductive formaldehydedimethylation have been widely used in mass-difference quantitationproteomics (Gygi et al., Nat. Biotechnol. 1999, 17:994; Li et al., Mol.Cell. Proteomics 2003, 2:1198-1204; Hansen et al., Mol. Cell. Proteomics2003, 2:299-314; Ong et al., Mol. Cell. Proteomics 2002, 1:376-386;Zhang et al., Anal. Chem. 2002, 74:3662-3669; and Hsu et al., Anal.Chem. 2003, 75:6843-6852).

Although being well-established methodologies for quantitativeproteomics, mass-difference labeling has two general limitations. First,typically only a binary set of samples can be compared due to the use oflight and heavy labeling of a peptide (although a triplex example cansometimes be obtained where light, medium and heavy isotopes are usedfor quantifying the samples). Second, mass-difference labeling increasesmass spectral complexity by introducing an extra pair of labeledpeptides, thus decreasing the confidence and accuracy of quantitation.The first limitation has been addressed and overcome by several researchgroups by introducing multiple heavy labeled reagents, rather than justone (Hsu et al., Electrophoresis 2006, 27:3652-3660; Morano et al.,Anal. Chem. 2008, 80:9298-9309; and Boersema et al., Proteomics 2008,8:4624-4632). However, the second limitation is an inherent drawback ofthe mass-difference approach, and the spectral complexity is onlyincreased with the use of multiple heavy isotope labeling reagents.

These limitations can be addressed through the use of isobaric labeling.There are two popular brands of isotopic labeling reagents, iTRAQ andTMT, currently sold in order to tag peptides when performingquantitative analysis of peptides using MS. Tandem mass tags (TMTs) werethe first isobaric labeling reagents used to improve the accuracy forpeptide and protein quantitation by simultaneous identification andrelative quantitation during tandem mass spectrometry (MS/MS or MS²)experiments (Thompson et al., Anal. Chem. 2003, 75:1895-1904). Twogenerations of TMTs were reported (TMT1 and TMT2), and each generationhad two isobaric labels. Amine groups (N-terminus and ε-amino group ofthe lysine side chain) in peptides labeled with TMT1 produce fragmentsat m/z 270 and 273 at 70 V collision energy, whereas TMT2 producesfragments at m/z 287 and 290 at 35 V collision energy. Relativequantitation can be performed by comparing the intensities of thesefragments to one another. A 6-plex version of TMTs was also recentlyreported (Dayon et al., Anal. Chem. 2008, 80:2921-2931), and 10-plex andeven 11-plex quantifications may be obtained using additional massdefect tags.

iTRAQ follows the same principle as TMTs quantitation, but it improvesthe quantitation further by providing four isobaric labels withsignature reporter ions that are one Da apart upon MS² fragmentation(Ross et al., Mol. Cell. Proteomics 2004, 3:1154-1169). Thus, iTRAQallows for the quantitation of proteins present in four differentbiological states simultaneously (a 4-plex quantitation). These tags arestructurally identical isobaric compounds with different isotopiccombinations. Each sample is labeled individually, pooled together, andintroduced into the mass spectrometer for quantitative analysis. Sincesamples are isobarically labeled, the same peptide from four samplesproduces a single peak in MS mode, but upon MS² fragmentation, eachlabeled sample gives rise to a unique reporter ion (m/z 114.1, 115.1,116.1, and 117.1) along with sequence-specific backbone cleavage foridentification. Relative quantitation is achieved by correlating therelative abundance of each reporter ion with its originating sample.

iTRAQ 8-plex quantitation follows the same quantitation principle asiTRAQ 4-plex quantitation (C. Leila, et al., Proteomics 2007, 7.3651-3660). Instead of using four reporter ions (m/z 114.1, 115.1,116.1, and 117.1) for quantitation of four samples, eight reporter ions(m/z 114.1, 115.1, 116.1, 117.1, 118.1, 119.1, 121.1 and 122.1) can beproduced and used for simultaneous quantitation of eight samples. iTRAQ8-plex reagents double the quantitation throughput over the 4-plexreagents. In addition to the higher throughput over a wider quantitationdynamic range, 8-plex reagents can also provide more accuratequantitation.

A common problem with the 4-plex iTRAQ reagent is that because the fourreporter ions are only one Dalton apart, isotope peak gains from orlosses to adjacent reporter ions affect both the accuracy and dynamicrange of quantitation for 4-plex samples. The quantitation accuracyproblem can be overcome by employing a complicated mathematic algorithmto quantify the reporter ions (A. Boehm, et al., BMC Bioinformatics2007, 8. 214). A software package is needed for 4-plex quantitationwhich brings extra cost for data analysis. The mathematic approach workswell for quantifying samples within ten-fold ratio difference. However,if two samples labeled by two adjacent reporter ions have abundancedifference greater than ten-fold, the reporter ion representing lowerconcentration sample could potentially be buried by the isotope peak ofthe adjacent reporter ion representing high concentration sample (S. Y.Ow, et al., J. Proteome Res. 2009, 8. 5347-5355). Therefore, thequantitation dynamic range is reduced. Two adjacent reporter ions withone Dalton mass difference should be avoided to quantify samples varyingin concentration greater than ten-fold. In this situation, 4-plexreagents can only be used to quantify two samples. 8-plex reagents canprovide two Dalton mass difference reporter ions for quantitation offour samples. Because of the minimal interference of adjacent reporterions, accurate quantitation and wider quantitation dynamic range can beachieved for four samples without sophisticated mathematical processing.Demand of high throughput protein/peptide LC/MS/MS quantitation inpractice makes iTRAQ 8-plex highly desirable for multiple samplequantitation. However, the trade-off of accurate quantitation and widerdynamic range is the high price of iTRAQ 8-plex reagents (about $2,500for a kit for five trials).

Isobaric MS² tagging approaches have also been successfully used inMS-based quantitative proteomics. However, their application as aroutine tool for quantitative MS studies is limited by high cost. Thehigh cost of commercial TMTs and iTRAQ comes from the challenge ofsynthesizing these compounds as multiple steps involved in the synthesislead to moderate to low yields. A set of 6-plex deuterium-labeled DiARTreagents was reported very recently with reduced cost of isobariclabeling. However, seven steps were still required to synthesize thesecompounds with only 30%-40% overall yield (Zeng et al., Chem. Commun.2009, 3369-3371). Additionally, many alternate MS labels are too labilewhich leads to cleaving the tag from the peptide of interest during massspectrometry analysis.

A new type of isobaric MS² tags with fewer steps involved in synthesisis desirable to further reduce experimental cost while taking fulltechnical advantages of the isobaric MS² tagging approach. Formaldehydedimethylation represents one of the most affordable approaches among allisotopic chemical derivatization techniques used for MS-based peptideand protein quantitation (Boersema et al., Proteomics 2008, 8:4624-4632;Ji et al., Proteome Res. 2005, 4:2099-2108; Ji et al., Proteome Res.2005, 4:1419-1426; Ji et al., Proteome Res. 2005, 4:734-742; Huang etal., Proteomics 2006, 6:1722-1734; Ji et al., Proteome Res. 2006,5:2567-2576; Ji et al., Anal. Chim. Acta 2007, 585:219-226; Guo et al.,Anal. Chem. 2007, 79:8631-8638; Wang et al., J. Proteome Res. 2009,8:3403-3414; Raijmakers et al., Mol. Cell. Proteomics 2008, 7:1755-1762;Synowsky et al., J. Mol. Biol. 2009, 385:1300-1313; Lemeer et al., Mol.Cell. Proteomics 2008, 7:2176-2187; Khidekel et al., Nat. Chem. Biol.2007, 3:339-348; Rogers et al., Proc. Natl. Acad. Sci. U.S.A. 2007,104:18520-18525; Aye et al., Mol. Cell. Proteomics 2009, 8:1016-1028;and Boersema et al., Nat. Protocols 2009, 4:484-494). However, isotopicformaldehyde labeling is a mass-difference labeling approach and, thus,lacks the advantages offered by the isobaric labeling approach.

A set of novel and cost effective N, N-dimethylated leucine (DiLeu)4-plex reagents were developed as an attractive alternative to iTRAQreagent for protein and peptide quantitation (Xiang, et al., Anal. Chem.2010, 82. 2817-2825). Additional isobaric tagging reagents containingamine reactive groups were also developed and described in U.S. Pat. No.9,388,132.

Isobaric Tandem Mass Tags Suitable for Carbohydrate and Glycan Labelling

Glycosylation refers to the process in which one or more of a widevariety of carbohydrates is attached to a functional group of anothermolecule, such as O-glycans and N-glycans attached to peptides (FIG. 1). Detection and quantification of glycans and glycosylation is highlyimportant as glycosylation is involved in several biological processesand abnormal glycosylation is involved in several diseases includingcancer, cardiovascular problems, and immunological disorders.Accordingly, intensive research efforts have been directed to massspectrometry (MS)-based quantitative glycomics.

A limiting factor of previous isobaric tagging reagents is that theywere designed to attach to the amine groups of peptides and are unableto react with carboxylic acid groups, or similar reactive groups,present in glycans. The present invention provides a set of novelisobaric chemical tags, also referred herein as SUGAR (IsobaricMultiplex Reagents for Carbonyl Containing Compound). These labelingtags are compact and easy to synthesize at high yield and purity in justa few steps using commercially available starting materials. Moreimportantly, the multiplex SUGAR tags are aldehyde-reactive,ketone-reactive and carboxylic acid-reactive, which offer the capabilityfor labeling and quantitation of glycans, proteins/peptides, and fattyacids (FIG. 2 ).

The tagging reagents of the present invention comprise: a) a reportergroup, having at least one atom that is optionally isotopically labeled;b) a balancing group, also having at least one atom that is optionallyisotopically labeled, and c) an aldehyde, ketone, or carboxylic acidreactive group able to react with an aldehyde, ketone, or carboxylicacid group of the molecule to be tagged, such as a glycosylated sidechain of a peptide.

A notable feature of this labeling approach is the production of intenseimmonium a1 ions when a dimethylated amino acid (such as a dimethylatedleucine) undergoes tandem mass spectrometry (MS²) dissociation.Additionally, the use of these tags enable multiplex analysis (i.e., atleast 4 plex, 8 plex, 12 plex, and even 16 plex analysis).

For 4-plex SUGAR tags, reporter ions with 1 Da mass difference in MS²spectra enable the use of mass spectrometers even with modestresolution, making it more broadly applicable to a wide variety ofinstrument platforms and accessible to a larger number of researchers.The performance of one of the SUGAR tags, hydrazide glycine dimethylleucine, has been benchmarked using both non-isotope version and 4-plexversion and have observed high labeling efficiency of glycans releasedfrom glycoprotein bovine thyroglobulin (>90% of all glycans, >95% ofaverage), enhanced backbone fragmentation at reduced normalizedcollision energies with improved intensity of reporter ions. Thisimproved performance will help to increase the accuracy and dynamicrange of glycan quantitation.

The tags of the present invention are beneficial in that they have thecapability to label both aldehyde, ketone, and carboxylic acidcontaining compounds. More specifically, the SUGAR tags arealdehyde-reactive, ketone-reactive, and carboxylic acid-reactive, whichoffer the capability for labeling and quantitation of glycans,proteins/peptides, and fatty acids. No commercially available tag hasbeen applied for aldehyde, ketone and carboxylic acid labeling.

SUGAR tags are also easy to synthesize at high yield and purity in justa few steps using commercially available starting materials, includingamino acids, formaldehyde, sodium cyanoborohydride, amino acids methylester, N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride,hydroxybenzotriazole, and hydrazine, thus providing an easy andcost-efficient synthesis approach.

The present tags also provide a simple labeling protocol and highlabeling efficiency. It only takes 2 hours to perform labeling chemistryfor SUGAR tags with glycans because of the high reactivity of hydrazidegroup. Additionally, high labeling efficiencies were observed forneutral and acidic N-glycans (>90%). Near complete labeling wereobserved for most of N-glycans, especially for some larger N-glycans,such as H₉N₂. (The labeling efficiency has been significantly improvedcompared to AminoxyTMT (˜50%), which is a commercially availableisobaric tag for quantitative glycan analysis.)

The present tags also provide high reporter ion yield with completebackbone fragment ions at a single collisional energy level. SUGAR tagsallow high reporter ion yield at a relatively low collisional energylevel, at which the backbone fragments were also preserved in a singlespectrum. Both the N-glycan backbone composition (backbone fragmentions) and the relative quantitation (reporter ion intensities) can beobtained in a single scan event. In contrast, difficulty is experiencedwhen using AminoxyTMT, which requires higher collisional energy torelease the reporter ion, thus, losing backbone fragment information. Inorder to obtain both structure and quantity information with AminoxyTMT,one glycan has to be fragmented with two different collisional energylevels: one at lower energy to obtain backbone fragments but minimalreporter ions; one at higher energy to obtain reporter ions but nobackbone fragments.

The present tags also provide good quantification performances (accuracyand precision) as well as high throughput multiplexed quantitativeanalysis. The quantification performance of SUGAR tags has beenevaluated by labeling N-glycans from glycoproteins and labeled with4-plex SUGAR tags at a ratio of 1:1:1:1. Low relative error (<15%) andsmall standard deviation were observed on various types of N-glycans(neutral/high mannose, neutral/complex, acidic/complex). Additionally,4-plex SUGAR tags can be synthesized at a fast and cost-effectivefashion, which significantly reduces instrument time and inter-samplevariance. Higher-plex versions of SUGAR tag are further possible withslight modifications.

SUGAR tags can also be applied on a wide variety of instrument platformswith MS/MS capabilities. It does not require special dissociationtechniques (ETD, ECD, etc.) which are only available on certaininstruments. Moreover, as 4-plex SUGAR is not a mass-defect based tag,it does not rely on high-resolution instrument platform. The reporterions with 1 Da difference in MS² spectra enable the use of instrumentwith low resolving power, making it more broadly accessible.

Additionally, the present invention provides isobaric 8-plex taggingreagents which can also be used to provide 16-plex reagents. In the caseof previous 4-plex reagents, the number of reporter ions was limited bythe balancing group, in that case a carbonyl group. Unfortunately, thecarbonyl group could only be modified 4 different ways using ¹³C and¹⁸O. To overcome this limitation, the present invention uses an aminoacid to form the balancing group. Amino acids were chosen for thebalancing group for several reasons: they can bear more isotopes than acarbonyl group, isotopic amino acids with various isotopic combinationsare readily commercially available, and the methods of coupling twoamino acids are well established.

With the general 8-plex reagent structure, instead of four reporter ionsfor four samples, eight reporter ions can be used to double thethroughput. Introducing 8 or more mass differences to the same analyteallows 8 or more different concentrations of the same analyte to bedetected in a single LC/MS run. The four reporter ions produced fromprevious 4-plex dimethyl leucine tagging reagents (m/z 115.1, 116.1,117.1, and 118.1) were extended to eight reporter ions (m/z 114.1,115.1, 116.1, 117.1, 118.1, 119.1, 121.1, and 122.1) by incorporatingdifferent numbers of stable isotopes (deuterium, ¹³C, ¹⁵N, and ¹⁸O) inan alanine based balancing group.

In addition, the 8-plex reagents provide increased quantitation accuracyand a wider quantitation dynamic range. A common problem forquantitation with 4-plex reagents using reporter ions which are only 1Da apart is the interference from adjacent isotopic peaks. Oftentimes, amathematic algorithm is needed to achieve accurate quantitation, as isthe case with the 4-plex reagents. In contrast, 8-plex and 16-plexreagents will offer a 2 Da separation between reporter ions if used in a4-plex manner. This mitigates the isotope interferences present with 1Da difference reporter ions thereby eliminating the need for complexmathematical processing in isobaric quantitative experiments.

Besides multiplex quantitation, the 8-plex and 16-plex dimethylatedamino acid tags also promote enhanced fragmentation, thereby allowingmore confident protein identification from tryptic peptides and de novosequencing of neuropeptides and metabolites.

The isobaric tagging reagents of the present invention can generally besynthesized in simple three or four step synthesis. The ease ofsynthesis provides high yields lowering the cost as compared to othercommercially available reagents. The ease of synthesis allows thesynthesis of tagging reagents with varying numbers of isotopes(primarily deuterium). Because each tagging reagent would produce areporter group having a unique molecular weight, each differentlylabeled molecule is able to be detected and relatively quantified bytandem mass spectrometry. It is believed using these different labels ina single experiment would act like multiple standards. The quantities oflabeled molecules can be calculated from a standard curve created usingthe different amounts of the different isotopically labeled molecules.Instead of having to run multiple different analyses, the sameinformation can be gleaned from a single experiment.

The development and application of a set of novel N,N-dimethylated aminoacid 8-plex, 12-plex, and 16-plex isobaric tandem mass (MS²) taggingreagents with high quantitation efficacy and greatly reduced cost forneuropeptide, protein and small molecule analysis are described below.These tagging reagents resemble the general structure of a tandem masstag in that it contains an aldehyde, ketone, or carboxylic acid reactivegroup, a balance group, and a reporter group. All labeling reagents arereadily synthesized from commercially available chemicals with greatlyreduced cost.

EXAMPLES Example 1—Isobaric Aldehyde-Reactive Dimethyl Leucines withImproved Reporter Ion Yield as Chemical Tools for Quantitative Glycomics

Glycosylation is one of the most important post-translationalmodifications as it is involved in several biological processes such ascell-cell recognition, communication and immunity response (Varki etal., Biological Roles of Glycans. In Essentials of Glycobiology, 2nded., Cold Spring Harbor (N.Y.), 2009; Moremen et al., Nat Rev Mol CellBiol, 2012, 13 (7): 448-62; Dwek, R. A., Chem Rev, 1996, 96 (2):683-720; and Defaus et al., Analyst, 2014, 139 (12): 2944-67). Abnormalglycosylation is relevant to diseases including cancer, cardiovascularproblems, neurodegenerative diseases, and immunological disorders(Taniguchi et al., Mol Cell Proteomics 2008, 7 (3): 626-7; An et al.,Curr Opin Chem Biol, 2009, 13 (5-6): 601-7; Alley et al., Chem Rev,2013, 113 (4): 2668-732; and Arnold et al., Proteomics, 2008, 8 (16):3284-93). Therefore, quantification of glycans is highly important andintensive research efforts have been directed to MS-based quantitativeglycomics. However, the difficulty in detection of native glycans limitsthe characterization and quantification because of the hydrophilicproperty, relatively low basicity as well as the lack of chromophore foroptical detection, resulting in poor ionization and detection using bothMS and optical methods.

Dimethyl leucine (DiLeu) is a set of isobaric tags originally designedand developed to have amine reactive groups for protein quantitativeanalysis (Xiang et al., Anal Chem 2010, 82 (7): 2817-25). Byincorporating ²H, ¹³C, and ¹⁵N isotopes into the DiLeu structure, up to12-plex DiLeu tags can be synthesized and utilized for simultaneousquantification of 12 samples or performing quantitative analysis oftriplicates of four different samples (Frost et al., Anal Chem, 2015, 87(3): 1646-54). Although DiLeu has recently become a powerful tool forquantitative proteomics, several challenges remain unsolved for use withglycans and similar molecules: (1) the difficulty in producingsufficient reporter ions during fragmentation for quantitation; (2) thedifficulty in obtaining glycan identification and relativequantification.

Several previous strategies have been developed to overcome the inherentlimitations of natural glycan detection. The most widely usedfluorescence detection (FIG. 3 ) was developed by Bigge et al. (AnalBiochem, 1995, 230 (2): 229-38). In this method, glycans were labeledwith 2-aminobenzamide (2-AB) or 2-aminobenzoic acid (2-AA) via reductiveamination to enable the fluorescence detection. 2-AB has since beenwidely applied in chromatographic analysis of glycans. The structuralassignment could be performed by comparing the elution positions withextensively developed database of 2-AB labeled glycans in hydrophilicinteraction liquid chromatography (HILIC) with fluorescence detection(Royle et al., Anal Biochem, 2008, 376 (1): 1-12). 2-AA is also widelyused in HPLC and CE separations for glycan analysis. It is a carboxylicacid which has capability to be detected in both positive and negativemode for neutral and sialic acid-rich glycan species (Anumula et al.,Glycobiology 1998, 8 (7): 685-94). Later, 2-aminopyridine (PA),1-aminopyrene-3,6,8-trisulfonic acid (APTS) and 2-aminonaphthalenetrisulfonic acid (ANTS) were well developed for different separation anddetection methods (Anumula et al., Anal Biochem 2006, 350 (1): 1-23).

With technological advancement of MS, MS-based quantitative glycomicshas become promising. The structural characterization as well asrelative quantification can be performed in a high-throughput manner.The native glycans can be labeled with hydrophobic small molecules thatcould increase the ionization efficiency. The backbone fragmentsproduced during fragmentation can be used to elucidate structure forcharacterization while intensities of parent ions or fragment peaksenable relative quantification of glycans.

There are three major strategies for relative quantification purpose.The most common strategy is isotopic labeling in which the targetmolecule is labeled with the same small molecule with different isotopeconfigurations. Different isotope configuration incorporates differentmass to the target. Thus, the relative quantification could be obtainedby comparing the intensities of the labeled glycan ions. For example,[H₄]-PA and [D₄]-PA labels were applied in the glycosylation analysis(Yuan et al., J Chromatogr A, 2005, 1067 (1-2): 145-52). Promisingrelative quantitative glycomics results were obtained by intensitycomparison of target ions with 4 Da difference. However, the retentiontime shift was observed between [H₄]-PA and [D₄]-PA labeled glycans on aC₁₈ column. To address the problem of retention time shift,[¹²C₆]-alanine and [¹³C₆]-alanine were used (Xia et al., Anal Biochem,2009, 387 (2): 162-70). No retention time shift was observed on C₁₈column. The other strategy performed separation of [H₄]-2-AA and[D₄]-2-AA labeled glycans by size exclusion to avoid the retention timeshift (Hitchcock et al., Proteomics, 2008, 8 (7): 1384-97). However,several disadvantages of isotopic labeling strategy are noted. First,the pairs of isotopic peaks add the complexity of the full MS scan,which could complicate spectral interpretation. Second, most isotopiclabels only contain dual plex that allow for binary quantitation. Sincethe mass difference between each plex should be greater than 4 Da toenable accurate quantification, the number of isotopes to beincorporated in labels imposes a limit on the plex number for multiplexquantitation using stable isotope labeling method.

Another labeling strategy is called mass defect labeling. Because of thedifferent nuclear binding energy for each atom, the ‘missing mass’ ormass defect for each atom is slightly different according to theEinstein's equation. The mass defect labeled glycan would have the samenominal mass while the accurate mass is slightly different. Thus, theintensity of pair of peaks could be used for relative quantificationusing high-resolution mass spectrometer. ¹³CH₃I and ¹²CH₂DI were used(Atwood et al., J Proteome Res, 2008, 7 (1): 367-74) in glycan analysisvia permethylation while 3 mDa difference was added by labeling witheither derivatization reagent for each permethylation site. Theadvantage of mass defect labeling is the ease of spectral interpretationsince the pair of peaks would merge together under low-resolution massmeasurement which also increases the detection sensitivity. However,high-resolution requires longer time for scanning process so that fewerspectra could be obtained during acquisition. The higher plex alsorequires ultra-high resolution, sometimes over 1 M resolving power isrequired for separating subtle mass differences between isobaric labels,which restricts the application of mass defect quantification.

Besides these two full MS-based quantification strategies, isobariclabeling becomes popular recently because of the simplified spectra,increased detection sensitivity, high plexing capability andlow-resolution requirement (Hahne et al., Anal Chem, 2012, 84 (8):3716-24; Yang et al., Sci Rep, 2015, 5: 17585; and Yang et al., AnalChem, 2013, 85 (17): 8188-95). The isobaric tags contain a reporter, abalancer and a reactive group. Different configurations of isotopes areincorporated into the reporter which can be fragmented to producereporter ions in low mass range for quantification purpose. The balancerhas another set of isotopes to keep the same nominal mass between eachtag. The reactive group is labeled with glycan to form the conjugate.Since each tag has the same nominal mass, the full MS spectra frommultiple samples remain relatively simple to interpret. Moreover, thelow-resolution requirement allows the profile to be acquired on mostinstrument platforms. The quantitative results could then be obtainedwith identification via backbone fragmentation. The intensities ofreporter ions can be used for quantification purpose.

Although several isobaric tags have been developed for glycan MSanalysis recently, the performances of these tags are limited. Forexample, AminoxyTMT (FIG. 4 ), which is a set of commercially availableisobaric tags for glycans, suffers from poor reporter ion yield and poorlabeling efficiency for some complex glycans. In general, the mostcommon labeling reaction for native glycan is reductive aminationbecause of the high specificity and complete labeling efficiency. Thelabeling condition for AminoxyTMT has been optimized decades ago (Biggeet al., Anal Biochem, 1995, 230 (2): 229-38). The concentration of thelabels was recommended to be at least 0.25 M while 1 M reducing agentwas required. The derivatization was performed in 30% acetic acid ofdimethyl sulfoxide within 2 hours. The reaction temperature of 60° C.was found to be optimal while higher temperature would accelerate thereaction with partial degradation and loss of sialic acid. However,AminoxyTMT performs labeling reaction via reversible imine formationwithout any reduction. Thus, it is possible for the conjugated glycan tomove back to the native glycan.

Additionally, if low collisional energy is used with AminoxyTMT, thereporter ion will not fully release. AminoxyTMT therefore requireshigher collisional energy to release the reporter ion; however, thiswill result in the fragmentation of the backbone fragment (FIG. 5 ). Inorder to obtain both structure and quantity information with AminoxyTMT,the glycan has to be fragmented with two different collisional energylevels: one at lower energy to obtain backbone fragments but minimalreporter ions; one at higher energy to obtain reporter ions but nobackbone fragments

QUANTITY (Yang et al., Sci Rep, 2015, 5: 17585) is another set ofisobaric tags for glycan analysis which contains quaternary amine withpermanent charge to improve the ionization efficiency. The labelingreaction yields near complete conjugated glycan via reductive aminationwhile the reporter ion yield is limited for some complex glycans becauseof the two cleavage sites of the reporter (FIG. 6 ). Lower energy couldonly produce limited reporter ions while higher energy would cleave thesecond site to diminish the intensity of reporter ion. The optimizedcollisional energy is critical for QUANTITY-labeled glycan moiety.

The successful development of high performance quantitative glycomicschemical tool provides an ideal workflow for quantitative glycananalysis in biological samples. Herein, the present example proposes aset of novel isobaric tags based on the customized DiLeu structure toovercome poor labeling efficiency and limited reporter ion yield bycombining DiLeu backbone with aldehyde-reactive group to create a highperformance quantitative glycomics chemical tool.

Candidate labeling tag structure design. DiLeu is a scaffold used withisobaric tags that was designed and developed for protein quantitativeanalysis almost a decade ago (Xiang et al., Anal Chem, 2010, 82 (7):2817-25). 12-plex DiLeu is available which can perform triplicatequantitative analysis of proteins from four different samples byincorporating ²H, ¹³C ¹⁵N isotopes into the DiLeu structure (Frost etal., Anal Chem, 2015, 87 (3): 1646-54). Although DiLeu has become apowerful tool for quantitative proteomics recently, several challengesremain unsolved: the difficulty in producing sufficient reporter ionsduring fragmentation and the difficulty in glycan identification andrelative quantification. However, by incorporating specific linkers andaldehyde reactive groups into the DiLeu structure, both challenges canbe solved. The isobaric tags for quantitative glycomics always sufferfrom the limited reporter ion yield while DiLeu backbone fragmentationrequires lower energy to produce reporter ions. In addition, thereactive group incorporated in the tag would increase the labelingactivity and enable simultaneous identification and quantification oflabeled glycan. Thus by combining DiLeu backbone with aldehyde-reactivegroup, a set of novel isobaric tags is provided to overcome poorlabeling efficiency and limited reporter ion yield.

As used in the examples below, “DiLeu” refers to a SUGAR tag based onN,N-dimethylated leucine having an aldehyde, ketone, and/or carboxylicacid reactive group. However it should be understood that SUGAR tags mayinclude compounds other than N,N-dimethylated leucine.

Preliminary results. The reactive group can be any functional group ableto react with any aldehyde, ketone, or carboxylic acid group of amolecule, including but not limited to a hydrazide, hydrazine, amine, oroxyamine (FIG. 7 ). As linkers could affect the fragmentation behaviorwhile reactive groups have different labeling activities, threecandidates were proposed (amine-DiLeu (A-DiLeu), hydrazide-DiLeu(H-DiLeu) and hydrazide-glycine-DiLeu (HG-DiLeu)) with different linkers(FIG. 8 ), including ethylenediamine, hydrazine and glycine; andreactive groups, including primary amine and hydrazide. With the DiLeubackbone structure, the reporter ions could be released with lowercollision energy. Either primary amine or hydrazide would allow regularreductive amination for labeling reaction to achieve high yield.

Synthesis strategy design. With the structures of three candidates beingdesigned, the synthetic strategies were subsequently developed. Becauseof the relatively simple functional group transformation, all thecandidates can be made within 3 steps from commercially availablematerials in high yield.

For A-DiLeu (FIG. 8 ), starting with leucine, reductive aminationreaction was applied to add dimethyl group onto the primary amine. Then,the mono-Boc-protected ethylenediamine was coupled with DiLeu to form aBoc-protected amide. In the last step, the Boc protecting group wasremoved by adding acid, like HCl in dioxane or trifluoroacetic acid(TFA) to produce desired A-DiLeu tag. The synthesis of H-DiLeu could bedone in 3 steps as well (FIG. 9 ). Leucine was treated with thionylchloride in methanol to convert carboxylic acid into methyl ester. Next,reductive amination added two methyl groups at the N-terminus ofleucine. In the final step, hydrazine was added to the dimethyl leucinemethyl ester in methanol or ethanol solution. The reaction was completedwithin 1 hour to convert ester to desired hydrazide DiLeu. However, asDiLeu reporter ion release undergoes amide cleavage, the hydrazide groupin H-DiLeu might affect the fragmentation behavior. HG-DiLeu wasdeveloped by employing glycine as linker between DiLeu and reactivehydrazide group to keep the amide cleavage possible (FIG. 10 ). Thesynthesis was done in 3 steps with 56% overall yield. Dimethylation infirst step is the same as regular DiLeu synthesis. Second, glycinemethyl ester was coupled with DiLeu by EDCl/HOBt amidation. Last,hydrazide was obtained from the methyl ester attacked by hydrazine inalmost quantitative yield.

Results. After the candidates were synthesized, the structure wasconfirmed by nuclear magnetic resonance spectroscopy (NMR) and MS, andthe candidates were aliquoted to 1 mg for performance evaluation werethe labeling efficiency, fragmentation behavior and quantitationaccuracy were examined.

The isotope atoms for the different isobaric tags can be placed atdimethyl group of N-terminus, N atom, carboxylic acid C atom as well aslinker positions. For example, isotope configuration of HG-DiLeu couldbe arranged as FIG. 11 shown for 4-plex isobaric tags. ¹⁵N, ¹³C-leucinewere used for HG-DiLeu 115 and 117 tags while the rest of two plex usednon-isotope leucine as starting material. For dimethylation step,D₂-formaldehyde was used for 118 tag to add 4 deuterium atoms at theN-terminus. Sodium cyanoborodeuteride was used for 116 and 117 tags toadd 2 deuterium atoms. The balancer was designed by using ¹⁸O atom atcarboxylic acid in the original 4-plex DiLeu. However, the 4 deuteriumatom difference between 115 and 118 tags produced several seconds LCretention time shift which could affect quantitation accuracy inisobaric labeling strategy. Thus, D₂-glycine methyl ester was used inthe 115 and 116 plex tags to decrease the deuterium number differenceand improve the quantitation performance. FIG. 11 only shows 4-plexHG-DiLeu synthesis. 12-plex, or higher plexes, is available by adding¹³C atoms at N-terminus with mass defect and high-resolution massspectrometer (FIG. 12 ). A 12-plex system is useful in that it enablestriplicate analysis of four different samples.

As the isotope-coded material is often expensive, the reaction yield iscritical for the tag synthesis. In the 3-step synthesis, the amidationstep might be a low yield reaction since the side chain of leucine wouldbe a steric issue which makes it difficult for EDCl to approach thecarboxylic acid as well as the glycine to attack the activated ester.However, the steric issue could be addressed with higher reactiontemperature, longer reaction time and stronger condensation reagents,such as PyAOP, HATU, DMTMM. Another potential pitfall is thefragmentation behavior change after connecting a linker. Since thefragmentation process often involves electron transfer, the linkers withelectron donating or electron withdrawing group were employed to alterfragmentation behavior.

Performance evaluation. After the tags were synthesized (¹H and ¹³C NMRof a SUGAR tag is shown in FIGS. 34 and 35 ), performance evaluation wasconducted to examine the labeling efficiency, fragmentation,quantitation accuracy, as well as ionization properties (FIGS. 13-26 ).Labeling efficiency is a very important performance metric to evaluatesince the complete yield of labeled glycan would provide moreinformation for characterization and less variation for the quantitationresult, while low labeling efficiency would limit the detection of lowabundance glycans. Also, the variable labeling yield could lead toinaccurate quantitation analysis. As the MS²-based quantitativeglycomics relies on the reporter ions produced via fragmentation, thefragmentation behavior is another important aspect to be evaluated. Inthis study, both labeling efficiency and fragmentation behavior wereexamined with linear (Glc)₈ as the standard.

(Glc)₈ was aliquoted to 100 ug for labeling and fragmentationperformance evaluation. The labeling reaction employed either imineformation for fast labeling or reductive amination for irreversiblelabeling. For imine formation labeling, the glycan standard was mixedwith 1 mg tag in 100 ul MeOH with 1% FA. The solvent was removed inSpeedVac after 10 min vortex. The labeling reaction was repeated twice.For reductive amination, the glycan standard was mixed with 1 mg tag in100 ul 30% acetic acid in DMSO containing 1 M NaBH₃CN. The labelingreaction was performed for 2 hours at 70° C. After the labeling, samplecleanup was carried out using 1 cc HLB Oasis cartridge. The cartridgewas conditioned with 3 ml 95% ACN, 1 ml 50% ACN and 3 ml 95% ACN. Then,the labeling solution was added in the cartridge filled with 1 ml 95%ACN. The cartridge was washed with 6 ml 95% ACN. The labeled glycan waseluted with 1 ml 50% ACN and 1 ml H₂O. The elution was dried in SpeedVacand stored in −20° C. for further use. The evaluation of labelingefficiency was carried out with a MALDI-linear ion trap-Orbitrap massspectrometer. The evaluation of fragmentation behavior was carried outusing a Q-Exactive Orbitrap mass spectrometer.

Both H-DiLeu and HG-DiLeu exhibited high labeling efficiency with imineformation which indicated the higher reactivity for hydrazide group.Over 85% yield could be obtained for either H-DiLeu or HG-DiLeucandidates with reductive amination while A-DiLeu could only produceless than 50% labeled glycan. With further development of the labelingstrategy, over 95% yield could be achieved for H-DiLeu and HG-DiLeu bystepwise reductive amination. The tag was mixed with glycan in MeOHcontaining 1% FA. The solvent was removed in SpeedVac after 10 minvortex. The labeling reaction was repeated twice. Then, 100 ul 30%acetic acid in DMSO containing 1 M NaBH₃CN was added into the tube andallowed to react for 2 hours at 70° C. The fragmentation behavior wasstudied for both hydrazide containing DiLeu candidates. The reporter ionwas only obtained from HG-DiLeu labeled glycans.

Evaluation of DiLeu tag candidates with glycoprotein standards. Afterthe performance evaluation with (Glc)₈ standard (FIGS. 13-16 ), HG-DiLeuwas selected as the best candidate while stepwise reductive aminationwas selected for the labeling reaction. The labeling efficiency,fragmentation behavior for several types of glycans and quantitativeaccuracy results were evaluated by labeling 4-plex HG-DiLeu withglycoprotein standards.

N-glycans were released from thyroglobulin from bovine thyroid (BTG)using the modified Filter Assisted N-Glycan Separation (FANGS) protocol(Abdul Rahman et al., J Proteome Res, 2014, 13 (3): 1167-76). HG-DiLeulabeling reaction was performed by reductive amination reaction. ForMALDI analysis, cotton HILIC SPE microtip was used for sample clean-upand data acquisition was carried out with a MALDI-linear iontrap-Orbitrap mass spectrometer. For ESI analysis, 1 cc Oasis HLBcartridge was used for sample clean-up and data acquisition wasperformed with a Q-Exactive Orbitrap mass spectrometer. Theidentification of glycans was performed by accurate mass matching at theMS¹ (i.e., full MS) level with fragmentation analysis at the MS² level.The relative quantitation between different samples was achieved bycomparing the intensities of the reporter.

With the stepwise reductive amination strategy, the glycans releasedfrom BTG were labeled with HG-DiLeu in high yield (FIG. 17 ). Thefragmentation produced relatively high intensity reporter ions forseveral types of glycans, especially, 11% comparing to the AminoxyTMTlabeled glycan which is only 2% intensity. For the quantitative accuracyexperiment, glycans released from the same amount of BTG were labeledwith different plex of HG-DiLeu. The preliminary quantitation resultsshowed relatively high accuracy with less than 15% relative error andlow standard deviation (FIG. 21 ).

Other ratios between different plex can be tested to further evaluatethe quantitation accuracy, such as 10:5:1:1 and 1:1:5:10. Also,different glycoproteins can be used to evaluate the performance of theHG-DiLeu tags with more types of glycans. Cells or human serum could beused for even more complex sample analysis. There are several potentialpitfalls including sialic acid loss during labeling reaction and limitedglycan release yield from complex samples. One strategy to reduce sialicacid loss is amidation for carboxylic acid. After the acid is convertedto amide, the stability is greatly enhanced. Since the sialic acid losshappens in acidic solution, recently developed reductive aminationlabeling condition could be used, such as methanol, ethanol andtetrahydrofuran (Anumula, K. R., Anal Biochem, 1994, 220 (2): 275-83;and Evangelista et al., Electrophoresis, 1996, 17 (2): 347-51). Ingeneral, glycans are released from glycoproteins with PNGaseF. However,the glycan release yield is not complete because the steric structuremakes it difficult for enzyme to access the glycosylation site. Complexglycoproteins could be digested with trypsin. Then PNGase F could beused to release glycans from glycopeptides to improve the overall glycanyield.

Example 2—Synthesis of Probes for Quantitative Glycomic Analysis

Materials and reagents. Methanol (MeOH), ethanol (EtOH), acetonitrile(ACN), dichloromethane (DCM), dimethyl sulfoxide (DMSO), acetic acid(AA), formic acid (FA) and water were purchased from Fisher Scientific(Pittsburgh, Pa.). Formaldehyde, sodium cyanoborohydride,N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCl),N-methylmorpholine (NMM), 1-Hydroxybenzotriazole hydrate (HOBt), glycinemethyl ester hydrochloride, hydrazine, triethylammonium bicarbonatebuffer (TEAB, 1.0 M), tris(2-carboxy-ethyl) phosphine hydrochloride(TCEP) were purchased from Sigma-Aldrich (St. Louis, Mo.). PNGase F waspurchased from Promega (Madison, Wis.). Oasis HLB 1 cc (30 mg)extraction cartridges were purchased from Waters Corporation (Milford,Mass.). Bovine thyroglobulin (BTG) was provided by Thermo FisherScientific (Rockford, Ill.). Microcon-30 kDa centrifugal filters (30KMWCO) were purchased from Merck Millipore Ltd. (Darmstadt, Germany).PolyGLYCOPLEX A™ beads (3 μm) were purchased from PolyLC Inc. (Columbia,Md.). Fused silica capillary tubing (inner diameter 75 μm, outerdiameter 375 μm) was purchased from Polymicro Technologies (Phoenix,Ariz.). All reagents were used without additional purification.

Synthesis of SUGAR tags. Formaldehyde (285 μL, 37% w/w) was added to astirred solution of sodium cyanoborohydride (120 mg) and L-leucine (100mg) in water (5 mL) at 0° C. After being stirred at room temperature for1 h, the reaction mixture was concentrated in vacuo. Purification of theresidue through column chromatography on silica gel (eluted withDCM/MeOH) afforded dimethyl leucine as a white solid.

Dimethyl leucine was dissolved in 10 mL DCM with 125 mg glycine methylester hydrochloride, 192 mg EDCl, 153 mg HOBt and 500 μL NMM. Afterbeing stirred at room temperature overnight, the reaction was purifiedwith a short column to remove solid byproduct. The crude residue wasconcentrated in vacuo and dissolved in 10 mL EtOH with 10% hydrazine.After being stirred for 3 hours at room temperature, the purificationthrough column chromatography on silica gel (eluted with DCM/MeOH)afforded SUGAR tag as a light-yellow oil (117 mg, 67% yield for 3steps). The 4-plex SUGAR tags were synthesized with heavy isotopeencoded starting materials (see FIG. 11 ).

¹H NMR (400 MHz, CDCl3): δ 0.93 (dd, J=9.2, 6.5 Hz, 6H), 1.50 (dddd,J=81.9, 13.4, 8.5, 5.3 Hz, 2H), 1.65-1.77 (m, 1H), 2.29 (s, 6H),2.94-3.05 (m, 1H), 3.71-4.04 (m, 4H), 7.74 (t, J=6.0, 1H), 8.28 (s, 1H).¹³C NMR (100 MHz, CDCl3): δ 22.1, 23.3, 25.9, 36.1, 41.6, 42.1, 66.8,170.0, 174.7.

Human serum protein preparation. The Health Sciences Institute ReviewBoard of the University of Wisconsin-Madison granted the permission toperform this study (2015-0864). Treatment was conducted according toprotocols AALL1131 or AALL0932. Serum samples from three B-cell acutelymphoblastic leukemia (ALL) pediatric patients were collected at thefollowing time points: before chemotherapy and at 1 month, 3 months, and6 months after the first day of consolidation chemotherapy. Theconcentration of serum protein was determined by microBCA assay.

N-glycan release by Filter-Aided N-Glycan Separation (FANGS). N-glycansof protein samples were released using FANGS with minor modifications(Abdul Rahman et al., J Proteome Res 2014, 13 (3): 1167-76). Briefly,protein samples were dissolved at a concentration of 1 μg/μL in 0.5 MTEAB buffer. TCEP (0.5 M, 5 μL) was added to the solution, which wasthen heat-denatured by switching sample tubes between 95° C. and roomtemperature water baths for 4 times (15 seconds each). A 30 K MWCOfilter was used to exchange 200 μL of 0.5 M TEAB buffer for 3 times. Theprepared protein samples on the MWCO filter were then incubated with 4μL PNGase F and 96 μL 0.5 M TEAB for 16 h at 37° C. water bath. Thereleased glycosylamines were separated from the de-glycosylated proteinsby centrifuging. Glycosylamines were collected into bottom tube and thede-glycosylated proteins remained above the filter. The filter waswashed with 100 μL 0.5 M TEAB buffer for reconstituting thede-glycosylated proteins. Both fractions were dried in vacuo. To convertglycosylamine to glycan (with free reducing end), 200 μL of 1% AA wasadded to the glycosylamine fraction, incubated for 4 h and dried invacuo.

N-glycan SUGAR labeling and cleaning up. SUGAR labeling reactions wereperformed using a stepwise strategy. Released N-glycans were mixed with1 mg SUGAR tag in 100 μL MeOH containing 1% FA. After 10 min incubation,the solvent was removed in vacuo. Then, 100 μL 1 M NaBH₃CN in DMSO: AA(7:3 v/v) was added to N-glycans. The reduction was performed at 70° C.for 2 h. The labeling reaction was cooled down before clean-up.

Oasis HLB 1 cc cartridge was used to remove excess labels and purify thelabeled N-glycans. The cartridge was conditioned with 1 mL of 95% ACN, 1mL of water, and 1 mL of 95% ACN. The crude reaction mixture was quicklyloaded to the conditioned cartridge which was pre-filled with 1 mL of95% ACN. The cartridge was then washed with 1 mL of 95% ACN for 3 times,and the labeled N-glycans were eluted with 1 mL 50% ACN and 1 mL water.The eluting fractions were combined, dried in vacuo, reconstituted in 50μL of 75% ACN, and analyzed by MALDI-MS or LC-MS/MS immediately.

MALDI-MS analysis for labeling efficiency calculation. Samples wereprepared by premixing 1 μL of SUGAR-labeled N-glycans with 1 μL2,5-dihydroxy benzoic acid matrix (150 mg/mL in 2% N, N-dimethylaniline,49% MeOH and 49% water), and 1 μL of each matrix/sample mixture wasspotted onto the MALDI target plate. A MALDI-LTQ-Orbitrap XL massspectrometer (Thermo Scientific, Bremen, Germany) was used forcharacterizing labeling efficiency. Ionization was performed using alaser energy of 15 μJ. Spectra were acquired in the Orbitrap massanalyzer within a mass range of m/z 1,000-4,000 at a mass resolution of30 K (at m/z 400).

LC-MS/MS analysis. A self-fabricated nano-HILIC column (15 cm, 75 μmi.d., 3 μm PolyGlycoPlex A HILIC beads) was used for glycan separation.A Dionex Ultimate 3000 nanoLC system was coupled to Q Exactive HF HybridQuadrupole-Orbitrap Mass Spectrometer (Thermo Scientific, Bremen,Germany) for all LC-MS/MS analyses. Mobile phase A was water with 0.1%FA, and mobile phase B was ACN with 0.1% FA. The flow rate was set at0.3 μL/min, and the injection volume was 2 μL. The following gradientwas used (time, % mobile phase B) unless otherwise specified: (0 min,75%), (18 min, 75%), (78 min, 15%), (78.1 min, 10%), (90 min, 10%),(90.1 min, 75%), (100 min, 75%).

The following MS parameters were used for all data acquisition in thisexample. Samples were ionized in positive ion mode with a spray voltageof 2 kV. S-lens RF level was set to be 55, and capillary temperature wasset to be 275° C. Full MS scans were acquired at m/z 500-2000 withresolving power of 30 K (at m/z 200). Maximum injection time of 100 ms,automatic gain control (AGC) target value of 1e6, and 1 microscan wereused for full MS scans. Top 20 data-dependent MS² analysis was performedat a resolving power of 15 K (at m/z 200) with collision-induceddissociation (CID) operating with a normalized collision energy of 30.The first mass was fixed at m/z 110, and dynamic exclusion of acquiredprecursors was set to 30 sec with a ±10 ppm tolerance.

N-glycan data analysis. SUGAR-labeled N-glycans were identified byaccurate mass matching. A peak list was exported and compared against anin-house database including most possible combinations of N-glycan units(Hexose (H), HexNAc (N), Fucose (F), and NeuAc (S)) with a masstolerance of 10 ppm. Reporter ion intensities in MS² were used forrelative quantification. Microsoft Excel was used for calculations andstatistical analyses.

Results and discussion. With the successful development of N,N-dimethylleucine tags (DiLeu) for quantitative proteomics by N-terminal labeling,the dimethyl leucine structure has been shown to produce abundantbackbone fragments with high-intensity reporter ions for quantitativeanalysis (Xiang et al., Anal Chem 2010, 82 (7): 2817-25; Frost et al.,Rapid Commun Mass Spectrom 2015, 29 (12): 1115-24; and Frost et al.,Anal Chem 2015, 87 (3): 1646-54). To implement conjugation with reducingend of glycans, the structure of SUGAR tags consists of a hydrazide asthe reactive group with glycine as a balancer. Using naturally-occurringamino acids as building blocks with straightforward functional grouptransformations, SUGAR tags can be synthesized in three steps (Scheme 4)with 67% overall yield:

In addition, the amino acid building blocks offer various commerciallyavailable isotope configurations to enable multiplexing capacity. Byincorporating different heavy isotopes, 4-plex SUGAR tags with reporterion mass difference of 1 Da can be readily synthesized and are shown inFIG. 28 . The multiplexing capacity can be further increased to 12-plexby employing mass defect concept (FIG. 31 ). However, the subtle massdifferences between reporter ions require higher resolving power. Futureefforts will demonstrate the utility of 12-plex tags for high throughputquantitative analysis.

Hydrazide chemistry is a commonly used conjugation for glycan reducingend labeling which converts hydrazide to hydrazone group. However, thereversible nature of hydrazide chemistry makes complete conjugationarduous. Thus, an alternative irreversible reductive amination is usedfor glycan reducing end labeling. Despite the high reactivity of thehydrazide group, a high concentration of a reducing agent, such as 1 MNaBH₃CN, can reduce glycans prior to hydrazone formation and yieldreduced glycan. To further improve labeling efficiency with minimalsample loss, a stepwise reductive amination has been developed.Maltooctaose standard was mixed with SUGAR tag in methanol with 1%formic acid for 10 minutes to complete hydrazone formation. Then,reduction was performed with NaBH₃CN in 7:3 (v/v) dimethylsulfoxide:acetic acid to achieve complete labeling (Scheme 5):

The stepwise reductive amination improved labeling efficiency bypreventing glycan reduction prior to hydrazone formation and reducedlabeling time by half (FIG. 32 ).

SUGAR tag with stepwise reductive amination was further applied to thelabeling of bovine thyroglobulin (BTG) standard. The workflow is shownin FIG. 27 . N-glycans released from BTG by PNGase F were labeled withSUGAR tags. Under optimized labeling conditions, SUGAR tags offer nearlycomplete labeling for different types of glycans including high mannose,complex/neutral, fucosylated, and acidic glycans. FIG. 17 highlightsseveral N-glycans labeling comparisons. The labeling efficiencies forother N-glycans are expected to be similar due to same shared corestructure of N-glycans. Labeling efficiencies shown in FIG. 18 arecalculated with following equation: labeling efficiency=labeled peakintensity/(labeled peak intensity+unlabeled peak intensity)×100%.

In order to maximize the fragmentation performance of SUGAR-labeledN-glycans, higher-energy collisional dissociation (HCD) performed withdifferent normalized collision energies (NCE) was examined. Mostbackbone fragments were observed with NCE of 20-25. The reporter ionintensities were elevated with minimal loss of backbone fragment ions byincreasing NCE to 30. At even higher NCE, the reporter ions became basepeak with a loss of the majority of backbone fragments. Thus, NCE of 30was chosen to yield high intensities of reporter ions along withabundant backbone fragments. The fragmentation behavior comparisonsagainst aminoxyTMT-labeled glycans are shown in FIGS. 19 and 20 .SUGAR-labeled N-glycans tend to produce higher intensities of reporterions and preserve more backbone fragments. Acidic N-glycans playimportant biological functions such as stability, degradation andantigenicity (Bork et al., J Pharm Sci 2009, 98 (10): 3499-508), thusattracting a great deal of research interests. However, acidic N-glycansoften produce fewer reporter ions than neutral N-glycans. With optimizedNCE, SUGAR-labeled acidic N-glycans produced more than a fourfoldincrease of reporter ion intensities upon fragmentation, demonstratingthe SUGAR tag's suitability for acidic N-glycan quantitative analysis.

Quantification performance of the 4-plex SUGAR tags was evaluated bylabeling N-glycans at known ratios. N-glycans released from BTG werealiquoted into four portions with known ratios at 1:1:1:1, 1:1:5:10 and10:5:1:1 in triplicate, then labeled with 4-plex SUGAR tags (see, forexample, FIG. 33 ). The intensities of reporter ions in MS² spectra foreach glycan were used to calculate the experimental ratios. In FIG. 29 ,panel A, experimental 4-plex SUGAR-labeled ratios for N-glycans areplotted against theoretical ratios 1:1:5:10. Representative MS² reporterions are shown in FIG. 29 , panels B-C for two SUGAR-labeled N-glycans.For all three known ratios, less than 15% relative errors are observedwith standard deviation of 0.2, 0.18 and 0.22, demonstrating that SUGARquantification approach offers accurate quantitative results.

As dynamic expression of N-glycans is highly relevant to biologicalprocesses, SUGAR tags were used to quantify N-glycans extracted from acomplex biological system at different biological states. Morespecifically, the SUGAR tags were used to analyze N-glycan changes inhuman serum proteins of Acute lymphoblastic leukemia (ALL) pediatricpatients (see Example 3 below).

Conclusions. In summary, new isobaric SUGAR tags with amino acidbuilding blocks were developed in this study with improvements in thefollowing aspects: low cost, high yield, complete labeling, highreporter ion yield, accurate and precise quantification, andapplicability for complex samples. Hydrazide reactive group enabledglycan reducing end conjugation while stepwise reductive aminationstrategy was developed to achieve complete glycan labeling. Thefragmentation of SUGAR-labeled glycans preserve more backbone fragmentswith higher reporter ion intensities for qualitative and accuratequantitative glycomics. SUGAR tags also show accurate quantificationwith broad dynamic range.

The successful development of SUGAR tags offers a useful chemical toolfor implementation in many biological and clinical applications. Thesimple building blocks, complete conjugation, desired fragmentation andaccurate quantification make it a precise contrivance for quantitativeglycomics study. It is anticipated that the novel SUGAR tagging approachcan be widely applied in multiple areas of biomedical research.

Example 3—Applications in Serum Analysis of Children ReceivingChemotherapy for B-Cell Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is one of the most predominantcancers for children which accounts for 26.8% childhood cancer diagnosesworldwide (Kaatsch et al., Cancer Treat Rev 2010, 36 (4): 277-85). Theuse of chemotherapy of the central nervous system has increased the5-year-event-free survival rate of around 80% in standard-risk ALL(Gaynon et al., Leukemia 2010, 24 (2): 285-97). However, it is reportedthat childhood cancer survivors suffer from neurobehavioral morbidityincluding diverse aspects of cognitive function, attention, processingspeed, memory, academic achievement, and emotional health, which has anegative impact on their quality of life (Speechley et al., J Clin Oncol2006, 24 (16): 2536-43). Previous studies indicated that theneurotoxicity of the chemotherapy could be revealed by alteration ofseveral protein expression levels (Krawczuk-Rybak et al., Adv Med Sci2012, 57 (2): 266-72). For example, Tau protein level in cerebrospinalfluid (CSF) serves as a biomarker of neuronal loss during activetreatment for ALL (Chen et al., Anal Chem 2018, 90 (13): 7817-7823).Glycans, play important roles in biological processes includingcell-cell recognition, communication and immunity response. Limited workhas been done on quantitative glycomics during chemotherapy. It isimportant to investigate the glycan level changes during the treatment,which could potentially facilitate biomarker discovery and lead toelucidation of pathogenesis mechanisms and discovery of potentialtherapeutic strategies and better treatments.

The present invention provides a set of isobaric tags based on thecustomized DiLeu structure to overcome these difficulties by combiningDiLeu backbone with aldehyde-reactive group to create a high performancequantitative glycomics chemical tool. The present isobaric tags wereapplied to quantitative glycomics of serum samples collected fromchildren receiving chemotherapy for B-cell ALL.

LC-MS/MS profiling of glycans released from serum samples of patients.Limited work has been done on quantitative glycomics during chemotherapytreatment. It is important to investigate the glycan level changesduring the cancer treatment given the aberrant glycan expression inseveral types of cancers, which will be useful for biomarker discoveryand identification of potential pathophysiological mechanisms.

N-glycans were released from serum samples collected from childrenreceiving chemotherapy using the modified Filter Assisted N-GlycanSeparation (FANGS) protocol (Abdul Rahman et al., J Proteome Res 2014,13 (3): 1167-76). HG-DiLeu (SUGAR tag) labeling reaction was performedby stepwise reductive amination. The identification of glycans wasperformed by accurate mass matching to the human serum glycan databasemanually at MS¹ level with fragmentation analysis at the MS² level. Therelative quantitation between different samples was achieved bycomparing the intensities of the reporter ions for each channel manually(FIG. 27 ).

N-glycan changes in human serum proteins of three B-cell ALL pediatricpatients were compared before induction and one month, three months, sixmonths after the chemotherapy. Four-plex SUGAR tags were used to labelN-glycans released from equal amounts of human serum proteins atdifferent time points. The same amount of protein was used for PNGase Fdigestion via FANGS protocol. The digested glycans were hydrolyzed in 1%acetic acid solution for 3 hours. Stepwise reductive amination wasemployed for the labeling process with different plex for samplecollected at different time point. After the clean-up with 1 cc HLBOasis cartridge, data was acquired on a Q-Exactive Orbitrap massspectrometer. Manual data analyses of three replicates provided glycanchanges during the chemotherapy. A 50% change was set as threshold forup-regulated or down-regulated changes. Since the amount of glycanmoiety was typically far less than protein, more serum samples should beused to collect enough glycans for quantitative analysis. The widespreadexistence of glycan isomers may require alternative separationmechanisms such as capillary electrophoresis, porous graphitic carbon(PGC) chromatography, or ion mobility separation, prior to MS analysisto achieve more accurate quantitative glycan analysis.

Quantitative analyses of selected N-glycans are summarized in FIG. 30with various types of N-glycans. Most quantified N-glycans reveal atrend of down-regulation after induction chemotherapy. In total, 145N-glycans were identified and quantified with SUGAR labeling approach.Of these, 68 N-glycans were quantified across all three patients. Theobserved down-regulated N-glycan expression could be explained byelimination of blasts after chemotherapy. As cancer cell metastaticgrowth can increase branching, fucosylation, and sialylation ofN-glycans (Norton et al., J Cell Biochem 2008, 104 (1): 136-49),chemotherapy could decrease such N-glycans by reverting this process.Indeed, the fucosylated and sialylated N-glycans show significantdown-regulation in patients after chemotherapy. The proof-of-principlestudy with SUGAR approach reveals the macroscopic relationship betweenchemotherapy and N-glycan expression. Further extensive and morein-depth investigations are needed to explore pathogenesis mechanismsand treatment outcomes.

Conclusion and significance. The 4-plex SUGAR tag quantitation approachwas applied to a complex biological system to investigate N-glycanchanges of B-cell ALL pediatric patients prior to and afterchemotherapy. It was found that most N-glycans were down-regulated afterchemotherapy, possibly due to cancer cell reduction.

Accordingly, the present SUGAR tags provide a novel chemical tool forglycan analysis and quantitative glycomics. The innovative stepwisereductive amination enables almost complete labeling of glycans in acomplex biological sample for detection of low abundance species. In themeantime, the high intensities of reporter ions produced viafragmentation, can benefit quantitative analysis of all types ofglycans. Additionally, 12-plex HG-DiLeu (SUGAR) is available by adding¹³C atoms at the N-terminus to enable triplicate analysis of fourdifferent samples in a single LC-MS/MS run. Therefore, these tags andmethods have broad impacts on both analytical tool development and itsapplication to biological and pharmacological investigations.

Example 4—Carboxylic Acid Labeling with SUGAR Tags

To label glycans through aldehyde and/or ketone groups (glycans), astepwise reductive amination is typically applied to achieve completelabeling as described above. To improve the labeling of carboxylicacids, organic amide coupling reagents such as EDCl, DCC, DIC, PyAOP,and HATU were used (see, for example, FIG. 2 ).

For example, carboxylic acid containing compounds were labeled withSUGAR tags by amidation. Twenty μg of carboxylic acid containingcompounds (peptides or fatty acids) were mixed with 1 mg SUGAR tag in100 μL DCM. EDCl was then added to a final concentration of 1 M. Thereaction was incubated for 2 h at room temperature. After solvent wasremoved in vacuo, 1 cc C₁₈ SPE was used to purify SUGAR-labeledcarboxylic acid containing compounds. The cartridge was conditioned with1 mL ACN, 1 mL of 50% ACN, and 1 mL water with 1% TFA. The crudereaction mixture was reconstituted in 1 mL water with 1% TFA and loadedto the conditioned cartridge. The cartridge was then washed three timeswith 1 mL water with 0.1% TFA, and the labeled peptides were eluted with1 mL 50% ACN with 0.1% FA and 1 mL 75% ACN with 0.1% FA. The elutionfractions were combined, dried in vacuo, and stored in −20° C. for MSanalysis.

The hydrazide group of SUGAR tags enables conjugation with bothaldehyde/ketone (glycans) and carboxylic acid (proteins/peptides andlipids) containing compounds. The fragmentations for different types ofbiomolecules are shown in FIG. 36 . Abundant backbone fragments enableisomer identification of biomolecules, while high intensity of reporterions (annotated with a star) can be utilized for quantification.

The SUGAR-labeled compounds demonstrated versatile conjugation abilitywith excellent fragmentation for qualitative and quantitativebiomolecule analysis. This feature makes SUGAR tags more distinct andunique compared to other commercial tags, as they are multi-functionalgroup reactive probes for a wide variety of biomolecules. This versatileprobe is to enable a broad spectrum of biomolecular quantitativeanalysis. An exemplary labeled glycan, peptide, and fatty acid are shownin FIG. 36 . FIGS. 37 and 38 further illustrate different steroidcompounds labeled with the SUGAR tags of the present invention.

Example 5—Mass Defect (Md)SUGAR for MS1-Based Glycan Quantitation andQuantitative Glycomics

Mass defect labeling was developed based on the slight mass difference(less than 1 Da) between different isotopes. The slightly different massof labeled compounds using this technique enables relativequantification of samples, provides easy spectral interpretation,increased detection sensitivity, and low abundance species quantitationcapacity. Most low abundance species might not be selected for MS²-basedfragmentation during the widely used data-dependent acquisition (DDA)with isobaric labeling. Since the quantitative results are obtained infull MS spectra, mass defect labeling enables the quantitative analysisfor low abundance glycans. With the development of robust and highresolving power instrument, the mass defect labeling is becoming apowerful tool for quantitative glycomics, and can be used as analternative to the MS²-based isobaric tag quantitative glycomicsstrategy. This example further provides a 3-plex mdSUGAR reagent for MS¹identification and quantification glycomics (see FIG. 39 ). Preliminarydata has demonstrated good performance (see FIG. 40 ) as well as thepotential to be expanded into 5-plex version corresponding with higherresolution mass spectrometers (i.e., 1,000K).

Having now fully described the present invention in some detail by wayof illustration and examples for purposes of clarity of understanding,it will be obvious to one of ordinary skill in the art that the same canbe performed by modifying or changing the invention within a wide andequivalent range of conditions, formulations and other parameterswithout affecting the scope of the invention or any specific embodimentthereof, and that such modifications or changes are intended to beencompassed within the scope of the appended claims.

When a group of materials, compositions, components or compounds isdisclosed herein, it is understood that all individual members of thosegroups and all subgroups thereof are disclosed separately. Everyformulation or combination of components described or exemplified hereincan be used to practice the invention, unless otherwise stated. Whenevera range is given in the specification, for example, a temperature range,a time range, or a composition range, all intermediate ranges andsubranges, as well as all individual values included in the ranges givenare intended to be included in the disclosure. Additionally, the endpoints in a given range are to be included within the range. In thedisclosure and the claims, “and/or” means additionally or alternatively.Moreover, any use of a term in the singular also encompasses pluralforms.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. Any recitation hereinof the term “comprising”, particularly in a description of components ofa composition or in a description of elements of a device, is understoodto encompass those compositions and methods consisting essentially ofand consisting of the recited components or elements.

One of ordinary skill in the art will appreciate that startingmaterials, device elements, analytical methods, mixtures andcombinations of components other than those specifically exemplified canbe employed in the practice of the invention without resort to undueexperimentation. All art-known functional equivalents, of any suchmaterials and methods are intended to be included in this invention. Theterms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed. The invention illustratively described hereinsuitably may be practiced in the absence of any element or elements,limitation or limitations which is not specifically disclosed herein.Headings are used herein for convenience only.

All publications referred to herein are incorporated herein to theextent not inconsistent herewith. Some references provided herein areincorporated by reference to provide details of additional uses of theinvention. All patents and publications mentioned in the specificationare indicative of the levels of skill of those skilled in the art towhich the invention pertains. References cited herein are incorporatedby reference herein in their entirety to indicate the state of the artas of their filing date and it is intended that this information can beemployed herein, if needed, to exclude specific embodiments that are inthe prior art.

We claim:
 1. A mass spectrometry tagging reagent comprising a compoundhaving the formula of:

wherein, R¹ is an aldehyde reactive group, ketone reactive group, orcarboxylic acid reactive group; R², R³, R⁴, R⁵ and R⁶, independently ofone another, are selected from the group consisting of hydrogen,deuterium, branched and unbranched C₁ to C₁₂ alkyl groups, C₄ to C₁₂cycloalkyl groups, C₂ to C₁₂ alkenyl groups, C₅ to C₁₂ cycloalkenylgroups, C₆ to C₁₂ aryl groups and C₇ to C₁₂ arylalkyl groups, whereineach of R², R³, R⁴, R⁵ and R⁶ optionally contain one or more ¹³C atomsand one or more deuterium atoms; C^(V) and C^(x), independently of oneanother, are ¹²C or ¹³C, O^(U) and O^(y), independently of one another,are ¹⁶O or ¹⁸O; and N^(z) and N^(W), independently of one another, are¹⁴N or ¹⁵N.
 2. The method of claim 1 wherein R¹ is a hydrazide,hydrazine, amine, or oxyamine.
 3. The tagging reagent of claim 1 whereinR² and R³, independently of one another, are CH₃, ¹³CH₃, CDH₂, ¹³CDH₂,CD₂H, ¹³CD₂H, CD₃ or ¹³CD₃.
 4. The tagging reagent of claim 1 wherein R⁶is hydrogen or deuterium.
 5. The tagging reagent of claim 1 wherein atleast one of: a) R² or R³ contains a deuterium atom, b) N^(z) is ¹⁵N, c)or N^(W) is ¹⁵N.
 6. The tagging reagent of claim 1 wherein R⁶ ishydrogen or deuterium and R⁵ is selected from the group consisting of:a) a methyl group containing one or more deuterium atoms and wherein thecarbon is ¹²C or ¹³C; b) hydrogen; c) deuterium; d) an isopropyl groupcontaining one or more deuterium atoms and one or more ¹³C atoms; and e)a butyl group containing one or more deuterium atoms and one or more ¹³Catoms.
 7. The tagging reagent of claim 1 wherein the tagging reagent hasa formula selected from the following:


8. A kit comprising two or more tagging reagents, wherein each taggingreagent comprises a reporter group, an aldehyde, ketone, or carboxylicacid reactive group, and a balancing group located between the reportergroup and aldehyde, ketone, or carboxylic acid reactive group, whereinone or more atoms in the reporter group, balancing group, or both, areisotopically heavy versions of the atom, wherein the reporter group ofeach tagging reagent has a mass different than the reporter groups ofthe other tagging reagents, the balancing group of each tagging reagenthas a mass different than the balancing groups of other taggingreagents, and the aggregate mass of the reporter group plus thebalancing group for each tagging reagent is the same; wherein thebalancing group of each tagging reagent has the formula:

wherein R⁵ and R⁶, independently from one another, are selected from thegroup consisting of hydrogen, deuterium, branched and unbranched C₁ toC₁₂ alkyl groups, C₄ to C₁₂ cycloalkyl groups, C₂ to C₁₂ alkenyl groups,C₅ to C₁₂ cycloalkenyl groups, C₆ to C₁₂ aryl groups and C to C₁₂arylalkyl groups, wherein each of R⁵ and R⁶ optionally contains one ormore ¹³C atoms and one or more deuterium atoms; C^(V) and C^(x),independently of one another, are ¹²C or ¹³C, O^(U) and O^(y),independently of one another, are ¹⁶O or ¹⁸O; N^(W) is ¹⁴N or 15N;wherein the reporter group of each tagging reagent has the formula:

R², R³ and R⁴, independently of one another, are selected from the groupconsisting of hydrogen, deuterium, branched and unbranched C₁ to C₁₂alkyl groups, C₄ to C₁₂ cycloalkyl groups, C₂ to C₁₂ alkenyl groups, C₅to C₁₂ cycloalkenyl groups, C₆ to C₁₂ aryl groups and C₇ to C₁₂arylalkyl groups, wherein each of R², R³ and R⁴ optionally contains oneor more ¹³C atoms and one or more deuterium atoms; and N^(z) is ¹⁴N or¹⁵N.
 9. The kit of claim 8 wherein the aldehyde, ketone, or carboxylicacid reactive group is a hydrazide, hydrazine, amine, or oxyamine. 10.The kit of claim 8 wherein R⁶ is hydrogen or deuterium.
 11. The kit ofclaim 8 wherein R² and R³, independently of one another, are CH₃, ¹³CH₃,CDH₂, ¹³CDH₂, CD₂H, ¹³CD₂H, CD₃ or ¹³CD₃.
 12. The kit of claim 8 whereinat least one of R² or R³ contains a deuterium atom, and N^(z) is 15 orN^(W) is ¹⁵N.
 13. The kit of claim 8 wherein the reporter group of eachtagging reagent has a mass that differs from one another by one or moreDaltons.
 14. The kit of claim 8 wherein the reporter group of eachtagging reagent has a mass that differs from one another by less than 25mDa.